bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic gains in oil yields after nine years of breeding Melaleuca alternifolia (Myrtaceae)
Authors: Doran J. C. , Baker G. R. , Williams E. R. , Southwell I. A.
Published: December 2006

A breeding program to enhance the economics of producing essential oil (Australian tea tree oil) from Melaleuca alternifolia plantations in eastern Australia by improving oil yields and oil quality commenced in 1993. This paper reports the results of the first (2003) and second harvests (2004) of a yield trial established by the program in 2002. The trial compares the performance of three grades of seed (clonal seed orchard, first generation seedling seed orchard and selected provenance) released to the industry by the breeding program against an unimproved seedlot (industry standard). Industry standard is representative of seed available to growers in 2001 from alternative sources within the industry. Progeny from the clonal seed orchard had the highest oil yield, giving an estimated average improvement in yield over industry standard of 83%. This was followed by selected provenance and first generation seedling seed orchard with 55% and 43% improvement, respectively. These yield improvements were accompanied by desirable changes in oil quality traits among breeding program seedlots which gave lower 1,8-cineole and higher terpinen-4-ol levels than industry standard. Economic analyses have demonstrated that, with current prices less than $20 per kg of oil, older plantations need to be replanted with new germplasm that will yield 90% more oil per hectare than plantings based on unselected seed if they are to deliver a 10% rate of return on investment. Gains of this magnitude are possible through the use of the breeding program’s clonal seed orchard seedlot. If the price drops further, then even greater yield gains will be necessary for farms to remain profitable. These gains will be delivered by the breeding program in the coming years through the release of seed from a second generation seedling seed orchard and fully tested clones suitable for mass propagation.
A decision framework for natural resource management: a case study using plant introductions
Authors: Wallace K. J.
Published: December 2006

One means of anticipating and, thus, preventing natural resource problems, such as those that may arise from plant introductions, is to use effective decision frameworks. This paper argues that such frameworks are typified by 4 elements. These are clear goals explicitly linked to cultural values, key questions that scope problems and management options, application of appropriate analytical tools, and the connection of authority for decisions with responsibility for outcomes. These elements are explored here. Trade offs are an inevitable part of decisions concerning natural resource management, including those relating to plant introductions. Benefit-cost and multi-criteria decision analyses are useful in this regard, but must be applied using methods that ensure all the relevant cultural values and management options are explored. Some recent proposals concerning the assessment of plant introductions do not always adequately frame decision issues. Ecological risk assessments can be used to define an acceptable level of risk concerning the negative impacts of introducing new biota, and, combined with an appropriate benefit-cost or multi-criteria analysis, provide the suite of analytical tools to make effective decisions concerning plant introductions. Effective decisions are more likely when the authority to make decisions and the responsibility for unforeseen outcomes are closely linked.
Inhibition of Cell Division by High External NaCl Concentrations in Synchronized Cultures of Chlorella emersonii
Authors: Setter T.L , Greenway H , Kuo J
Published: December 2006

Effects of high external NaCl concentrations on growth were examined in the unicellular freshwater
alga Cldorella emevsonii during different phases of cell development, using synchronized cultures
obtained by alternating light-dark cycles. Growth of cultures synchronized at 1 mM NaCl [external
osmotic pressure (next=) 0.08 MPa] was compared with (i) cultures synchronized at 200 mM NaCl
(n,,, = 1.01 MPa) and (ii) cultures synchronized at 1 mM NaCl from which the daughter cells were
suddenly transferred to 100, 150 or 200 mM NaCl. The effects of these two treatments on synthesis
of protein, RNA and DNA during cell cycles were similar, and are attributed to the high nexta nd
not to specific effects of Na+ and C1-.
Growth inhibitions in cells at 200 mM NaCl relative to 1 mM NaCl occurred mainly via effects
on cell division; this was confirmed by electron microscopy. There was a lag before net DNA
synthesis commenced, and there were reductions in rates of net DNA synthesis in cells at 200 mM NaCl
relative to 1 mM NaC1. Rates of increase in cell volume and in protein and RNA content per cell
were little affected by high external NaCl concentrations. Consequently, daughter cells at 200 mM
NaCl were approximately twice the volume and contained twice as much protein and RNA as
daughter cells at 1 mM NaCl, while DNA content was equal in daughter cells at 1 and 200 mM NaCl.
Cell Division and Differentiation in Cambial Explants of Carrot-root Tissue
Authors: Lancaster D.M , Rowan K.S
Published: December 2006

Cambial explants of carrot-root were grown in axenic culture without added hormones. The cambial
initials neither retained their identity nor continued to generate xylem and phloem elements. Cell
division and differentiation proceeded in newly initiated peripheral cambia. Indol-3-yl acetic acid,
2,3,5-triiodobenzoic acid and kinetin, when added to the medium, did not retain the original cambial
function but induced essentially similar differentiation. We conclude that active cell division was not an
innate attribute of cambial initial cells and the mobile material they contained activated cell division in
other somatic cells.
Evidence for Osmotic Adjustment in Phalaris tuberosa L. cvv. Australian and Sirosa
Authors: Sambo E.Y , Aston M.J
Published: December 2006

The temperate pasture grass Phalaris tuberosa L. (cvv. Australian and Sirosa) was grown in soil under glasshouse conditions. When water was withheld, leaf xylem potential (�) decreased at the rate of 0.02 and 0.05 MPa per day in cvv. Australian and Sirosa, respectively, between 7 and 17 days, and reached a dawn value -0.25 (� 0.02) MPa and -0.56 (�0.08) MPa in the respective cultivars. These plants were moderately stressed. Between 17 and 23 days, when the experiment was terminated, stress developed more rapidly and � at dawn reached final values of -2.1 (�0.09) and -2.2 (�0.08) MPa in Australian and Sirosa phalaris, respectively. These plants were severely stressed.
The leaf osmotic potential (��) decreased at similar rates as � in the stressed plants, thus maintaining the turgor potential (�*p) relatively constant with increasing water stress. Osmotic adjustment (��100/�) was judged by comparing �� at full turgor (�100/�) in stressed plants which had been rewetted, with �100/� of control unstressed plants. ��100/� of moderately stressed plants was 0.46 and 0.48 MPa in Australian and Sirosa phalaris, respectively. In severely stressed plants, the respective ��100/� values were 0.67 and 0.85 MPa.
The Vascular System of the Wheat Spikelet
Authors: O'Brien T.P , Sammut M.E , Lee J.W , Smart M.G
Published: December 2006

The attachment region of a mid-spike spikelet was sectioned serially. These sections were used to
construct an accurate 3-dimensional model of the course of the vascular system that supplies the organs
of the a and b florets, and the rachilla of the c and d florets. All organs are interconnected by vascular
tissue, but certain parts of the system are phloem-only. In particular, the supply to the groove bundle
of the pericarp, widely held to be the most important pathway to the grain, is made via an annulus of
phloem to which lemma, palea and lodicules have phloem-only connections. The vascular system is
sufficiently different from the pattern encountered in vegetative nodes to warrant treatment sui generis.
The relationships between different cell types need greater histological study, especially in the complex
composite bundles. This analysis shows that bundle shape in cross-section and the arrangement
of xylem and phloem vary sharply over very short distances (100 ~ m ) .T he distribution of xylem and
phloem transfer cells agrees with the proposal that significant solute relocation takes place in the regions
where the vascular supplies to different organs meet. The area in the ovary neck that encompasses the
fusion zone of the supplies to lemma, palea and pericarp emerges as a zone in need of detailed study,
both in spikelet positions within a cultivar of known, but different, grain performance, and as a region
to analyse for inter-cultivar comparisons.
Dry storage of sperm: applications in primates and domestic animals
Authors: Meyers Stuart A.
Published: December 2006

Cryopreservation of spermatozoa, oocytes and embryos, as well as somatic cells or cell lines for cloning from cells, are all options for the long-term storage of unique genotypes and endangered species. Spermatozoal cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration-based methods for long- or short-term storage, which requires routine maintenance and extensive space requirements. The preservation of stem cells also has strict requirements for long-term storage to maintain genetic integrity. Dessicated (lyopreserved) sperm and stem cells will provide an unprecedented type of long-term storage without the need for expensive and burdensome cryogenic conditions. Experiments were conducted to determine an effective intracellular concentration of the lyoprotectant trehalose. High-pressure liquid chromatography studies revealed that trehalose can be incorporated into mature sperm cells as well as spermatogonial stem cells from rhesus monkeys. In addition, using fourier transform infrared spectroscopy, we determined that thermotropic phase transitions for fresh ejaculates from rhesus monkey and stallion sperm occurred at 10–15, 33–37 and 55–59°C. Preliminary studies in our laboratory have indicated that spermatogonial stem cells can be dried to <3 g g−1 water and maintain viability following rehydration. Studies in our laboratory have provided preliminary results suggesting that the desiccated storage of sperm and spermatogonial stem cells may be a viable alternative to conventional cryopreservation.
Gamete imprinting: setting epigenetic patterns for the next generation
Authors: Trasler Jacquetta M.
Published: December 2006

The acquisition of genomic DNA methylation patterns, including those important for development, begins in the germ line. In particular, imprinted genes are differentially marked in the developing male and female germ cells to ensure parent-of-origin-specific expression in the offspring. Abnormalities in imprints are associated with perturbations in growth, placental function, neurobehavioural processes and carcinogenesis. Based, for the most part, on data from the well-characterised mouse model, the present review will describe recent studies on the timing and mechanisms underlying the acquisition and maintenance of DNA methylation patterns in gametes and early embryos, as well as the consequences of altering these patterns.
Spermatogonia: origin, physiology and prospects for conservation and manipulation of the male germ line
Authors: Ehmcke Jens , Hübner Karin , Schöler Hans R. , Schlatt Stefan
Published: December 2006

In recent years, the scientific community has become increasingly interested in spermatogonia. Methodological breakthroughs, such as germ cell transplantation and spermatogonial culture combined with novel germ line transfection strategies, have provided interesting new opportunities for studying the physiology of spermatogonial stem cells and their interaction with the stem cell niche. Furthermore, intense research into pluripotent and adult stem cells has generated new insight into the differentiation pathway of germ line stem cells and has opened new perspectives for stem cell technologies. The present review briefly introduces the physiology of spermatogonial stem cells and discusses future directions of basic research and practical approaches applicable to livestock maintenance and animal reproduction.
Placental function in development and disease
Authors: Cross James C.
Published: December 2006

The placenta is an organ that clinicians and embryologists would all agree is important for pregnancy success. Unfortunately, however, they too often ignore it when they are exploring causes for embryonic, fetal and perinatal complications. The core function of the placenta is to mediate the transport of nutrients between the maternal and fetal circulation, but it also has critical endocrine functions that alter different maternal physiological systems in order to sustain pregnancy. Both its development and ongoing functions can be dynamically regulated by environmental factors, including nutrient status and tissue oxygenation. In recent years, mainstream attention has begun to shift onto the placenta and it is now becoming clear that placental pathology is associated with several complications in human and animal pregnancies, including embryonic lethality, fetal growth restriction, pre-eclampsia and the high rates of fetal deaths observed after nuclear transfer (cloning).
Male germ cell transplantation in livestock
Authors: Hill J. R. , Dobrinski I.
Published: December 2006

Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production.
Oocyte cryopreservation: oocyte assessment and strategies for improving survival
Authors: Ledda Sergio , Bogliolo Luisa , Succu Sara , Ariu Federica , Bebbere Daniela , Leoni Giovanni Giuseppe , Naitana Salvatore
Published: December 2006

Despite significant progress in cryopreservation of mammalian oocytes and embryos, many of the molecular and biochemical events that underlie this technology are poorly understood. In recent years, researchers have focused on obtaining viable oocytes that are developmentally competent. Even under the most favourable conditions, experimental approaches have achieved only limited success compared with fresh oocytes used in routine in vitro embryo production. Chilling injuries and toxic effects of the cryoprotectants are the major adverse consequences following cryoprocedures. To overcome these problems, different strategies have been developed for improving cryopreservation results. These strategies include reducing container volumes, increasing the thermal gradient, changing the cell surface/volume ratio, enhancing cryotolerance by supplementation with various additives or modifying the lipid composition of the oocyte membrane. In order to develop new strategies for reducing the various forms of stress associated with oocyte cryopreservation, it is fundamental to gain a better understanding of the major changes responsible for poor post-thaw survival. With this knowledge, we hope that oocyte cryostorage will become a fully reliable reproductive technique in the near future.
10 PREGNANCY RATE IN DAIRY COWS CROSSBREED HOLSTEIN × CARORA, DURING TWO SEASONS OF THE YEAR UNDER TWO ESTRUS SYNCHRONIZATION PROGRAMS AND TROPICAL CONDITIONS IN VENEZUELA
Authors: Cabezas M. , Cardenas J. , Diaz T. , Kowalski A. A.
Published: December 2006

Two experiments were performed in a commercial farm located in Lara, Venezuela. The objective was to evaluate the pregnancy rate in postpartum dairy cows with different genetic composition of Holstein (H) and Carora (C) breeds (1/2, 3/4, and 7/8) during two seasons of the year using estrus synchronization programs. The first experiment evaluated the pregnancy rate of three different breeding groups in two seasons of the year. All cows (n = 305) were treated with the Heatsynch program (Select Sires, Inc., Miami, FL, USA): Cycling cows between Days 36 and 42 after parturition were injected with two doses of PGF2� 14 days apart. Seven days later, after the last injection of PGF2�, the cows were injected with a GnRH analog (Buserelin). Seven days later cows were injected with PGF2� and after 24 h cows were injected with 1 mg of estradiol cypionate (ECP). Cows were inseminated when heat was detected, and after 48 h all cows that did not present heat were inseminated. During the period from January to May, 152 cows were treated. The average temperature (T) was 25.22�C and relative humidity (RH) was 70.16%. During the period from June to October, 153 cows were treated. The T was 25.26�C and RH was 72.58%. The pregnancy rates for the groups were 1/2H � C, n = 89, 28.08%; 3/4H � C, n = 143, 25.17%; and 7/8H � C, n = 73, 26.02%; they were not significantly different (P < 0.14). However, the pregnancy rate was higher in January-May (30.92%) than in June-October (22.87%; P < 0.05). The second experiment evaluated pregnancy rates in the different breeding groups (H � C), using two protocols, Heatsynch vs. Ovsynch (Select Sires, Inc.). The Ovsynch program is similar to the Heatsynch program; however, the last injection of ECP in the Heatsynch program is changed to the GnRH analog after 48 h from the last injection of PGF2�. Cows were inseminated 24 h after the GnRH injection. Pregnancy rate was lower for cows treated with the Heatsynch program (21.15%) than for those treated with the Ovsynch program (45.28%; P < 0.01). Furthermore, cows in the 1/2 and 3/4 breeding groups had higher pregnancy rates than those in the 7/8 breeding group (1/2 = 42.05%, 3/4 = 38.33%, and 7/8 = 16.72%; P < 0.01). Data were analyzed using logistic regression analysis. In conclusion, the introduction of the Carora breed in the dairy herd increased the adaptability of the Holstein cows to tropical conditions by increasing their reproductive efficiency. Furthermore, pregnancy rates were increased using synchronization programs that did not require heat detection. This demonstrated the possibility of increasing reproductive performance in the tropics by incorporating estrus synchronization systems that do not require heat detection. These studies also demonstrated that the incorporation of dairy breeds adapted to tropical conditions, such as the Carora breed, aids in improving reproductive performance under high RH conditions.
100 TIMING OF BLASTOCOELIC CAVITY RE-EXPANSION REPRESENTS A QUALITY INDEX OF IN VITRO PRODUCED OVINE BLASTOCYSTS
Authors: Leoni G. G. , Berlinguer F. , Succu S. , Mossa F. , Galioto M. , Bebbere D. , Naitana S.
Published: December 2006

There is general consensus that the quality of in vitro-produced embryos is inferior to that of their in vivo counterparts and that along with other factors this may be responsible for the poorer cryosurvival and the lower pregnancy rates reported for these embryos. The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess whether in vitro-produced ovine embryo quality could be determined by the timing blastocoelic cavity re-expansion after vitrification, thawing, and in vitro culture. Embryos were produced in vitro from ovine ovaries collected at the local slaughterhouse following a standard protocol developed for ovine oocytes, as previously described (Berlinguer et al. 2005 Reprod. Fertil. Dev. 17, 201). Blastocysts were then vitrified/warmed according to a simple method (Leoni et al. 2002 Cryobiology 45, 204-212) and cultured in vitro in TCM-199 supplemented with 10% FCS for 72 h in 5% CO2 in air at 39�C. Vitrified embryos were divided into two groups: (A) expanded within 8 h of in vitro culture after warming; (B) expanded during 8 to 16 h of in vitro culture after warming. Of the 338 vitrified/warmed embryos, 173 (51.1%) showed a re-expanded blastocoelic cavity after 8 h of in vitro culture, whereas 58 (17.1%) re-expanded during 8 to 16 h of in vitro culture. We also analyzed by semiquantitative RT-PCR a panel of genes expressed during pre-implantation embryo development (glucose transporter, HPS-10, e-caderin, poly(A)polimerase, and ubiquitine); our results showed that these genes, apart from ubiquitine which showed no difference in the two groups, were more expressed in Group A blastocysts compared to Group B. Group A blastocysts showed higher hatching rates (67.6%) than Group B blastocysts (43.1%; P < 0.01). The total cell number calculated for the hatched blastocysts after staining with Hoechst 33342 was significantly higher in Group A (140.7 � 8.3, n = 42) than Group B (102.2 � 8.4, n = 27; P < 0.01). Pregnancy rate (detected by ultrasonography 30 days after embryo transfer) after laparoscopic embryo transfer to 12 synchronized recipient Sarda sheep was 40% (6/15) in Group A and 13.4% (2/15) in Group B. The results indicated that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro-produced ovine embryo quality, and in addition, a better prediction for improved survival rate after transfer to synchronized recipients.
This work was supported by Cofin 2003.
105 COMPARISON BETWEEN TWO EXTENDERS FOR CRYOPRESERVATION OF BACTRIAN CAMEL SEMEN
Authors: Niasari-Naslaji A. , Mosaferi S. , Gharahdaghi A. A. , Abarghani A. , Ghanbari A. , Gerami A. , Bahmani N.
Published: December 2006

A Tris-based extender (SHOTOR diluent) has been developed for preserving Bactrian camel semen at 4�C (Niasari-Naslaji et al. 2005 Reprod. Fertil. Dev. 17, 198 (abstr.)). The present study investigated the possibility of utilizing the SHOTOR diluent for the cryopreservation of Bactrian camel semen. A modified bovine artificial vagina (Masaferi et al. 2005 Theriogeology 63, 92-101) was used to collect semen from three fertile bulls. The viscosity of the semen was reduced mechanically (Mosateri et al. 2005) and the homogenized semen was divided equally into two parts. Each part was sequentially diluted with either IMV buffers (Green buffer: first extender; White buffer: second extender; IMV, France) or SHOTOR diluents (without glycerol: first extender; with 12% glycerol: second extender). SHOTOR diluent consists of 2.6 g TIS, 1.35 g citric acid, 1.2 g glucose, and 0.9 g fructose in 100 mL of deionized water, with an osmolality of 330 mOsm/kg and pH of 6.9. All extenders had 20% egg yolk and antibiotics. The semen was diluted at the ratio of 1:1 with the first extender. The diluted semen was then cooled within 2 h to 4�C. At this temperature, the second extender was added at the same volume as the diluted semen in three steps with an equal volume, 10 min apart. After a 30-min equilibration time, beginning after addition of the last fraction of the second extender, the diluted semen was loaded into 0.5-mL straws at a concentration of 50 � 106 sperm per straw. The straws were maintained for 20 min at 4 cm above the liquid nitrogen surface, after which they were plunged into liquid nitrogen. The semen was thawed at 40�C water bath for 20 s. Progressive forward motility of spermatozoa was assessed at the time of dilution and immediately after thawing of the semen. The experiment was replicated four times. Data were analyzed using GLM procedure in SAS/STAT after arcsine transformation. At the time of dilution, there was no significant difference in progressive forward motility of spermatozoa between IMV buffers (51.8%) and SHOTOR diluent (61%; P > 0.05). However, after thawing, there was a significant decrease in progressive forward motility of spermatozoa in IMV buffers (4.2%) compared to SHOTOR diluent (29.9%, P < 0.05). In conclusion, in this experiment, SHOTOR diluent was more efficient for cryopreserving Bactrian camel semen than IMV extender.
Shotor means camel in the Persian language.
106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS
Authors: Nicacio A. C. , Simões R. , Peres M. A. , Gonçalves J. S. A. , D'Ávila Assumpção M. E. O. , Visintin J. A.
Published: December 2006

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability.
This work was supported by FAPESP 04/05335-1.
108 NEGLIGIBLE LEVEL OF DNA DAMAGE IN BOVINE OOCYTES VITRIFIED USING MINIMUM VOLUME METHODS
Authors: Papis K. , Stachowiak E. , Kruszewski M. , Iwanenko T. , Bartlomiejczyk T.
Published: December 2006

A relatively high number of bovine cryopreserved oocytes analyzed by the comet assay (Men et al. 2003 Mol. Reprod. Dev. 64, 245) showed compromised DNA integrity. The DNA fragmentation (comet tails) was found in 29% of slow cooled oocytes, in 20% of oocytes vitrified in straws and in 24% of oocytes vitrified in open pulled straws (OPS). Present study used the comet assay to compare the DNA status of 151 in vitro matured bovine oocytes vitrified in straws, in OPS or in droplets. It was assumed that the droplet method (Papis et al. 2000 Theriogenology 54, 651), which has gentle pre-equilibration prior to vitrification, would offer better protection of DNA. OPS vitrification was performed using a solution consisting of 20% DMSO, 20% ethylene glycol (EG), and 0.5 M sucrose. For in-straw and in-droplet vitrification, VS14 (5.5 M EG and 1.0 M sucrose) solution was used. In these two methods pre-equilibration in 3% EG solution for 15 min was applied. Fresh oocytes exposed to 0.5 mM of hydrogen peroxide for 5 min served as the positive control. Fresh M II oocytes served as the negative control. The comet assay was performed according to the procedure of Men et al. (2003) with some modifications aimed at enhancing the sensitivity of the method. The zona pellucida was removed using 0.5% pronase solution, followed by placing of the oocytes in droplets of low-melting agarose on slides. These were subjected to overnight treatment in lysis buffer, followed by 40 min of DNA unfolding and 30 min electrophoresis. Following air drying, the slides were stained with DAPI fluorochrome and photographed. The pictures were saved as anonymous consecutive files to enable objective assessment. Of 119 vitrified oocytes, 112 (94%) were evaluated. The remainder were lost or displayed atypical pictures. The comets could not be analyzed with the Comet v.3.0 software, possibly due to the large size of each oocyte. Six main classes of comet tails were distinguished ranging from no tail (class 0) to heavy and long tail (class 5). Positive control oocytes displayed class 4 (36%) or 5 (64%) tails. Negative control oocytes formed class 0 (18%) to class 3 (47%) comet tails. The oocytes vitrified using minimum volume methods fell within the same range, with 80% and 76% of oocytes vitrified in droplets and OPS, respectively, forming class 1 or 2 tails. One OPS vitrified oocyte (2.2%) expressed a class 5 tail. A small but significant proportion of oocytes vitrified in straws (15.4%, P d 0.05, ANOVA) formed class 4 tails typical of positive control oocytes. In conclusion, in spite of pre-equilibration, a significant proportion of oocytes vitrified in straws contained detectable levels of DNA fragmentation, due probably to the lower cooling rate. The minimum volume protocols (the droplet and OPS methods) caused virtually no damage as assessed by the DNA comet assay. Results presented here differ from those reported previously. Reasons for differences remain to be established.
113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA
Authors: Thys M. , Van Soom A. , Dewulf J. , Rijsselaere T. , de Kruif A.
Published: December 2006

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing.

Table 1.
VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm

This research was supported by IWT (no. IWT/020727).
117 VITRIFICATION OF CHAROLAIS AND HOLSTEIN BLASTOCYSTS USING LN SLUSH
Authors: Yavin S. , Ditesheim D. , Zeron Y. , Arav A.
Published: December 2006

Cryopreservation of embryos for transfer in outdoor conditions is a challenge; therefore a rapid and simple technique should be applied. Two different experiments were performed to develop a functional and efficient vitrification technique. Charolais (for Exp. 1) and Holstein (for Exp. 2) female cows were superovulated and artificially inseminated (AI). Seven days after AI, embryos were flushed from the uterus and vitrified. For vitrification, blastocyst were exposed to 10% vitrification solution (VS) for 3 min, transferred into 50% VS, and immediately thereafter into 87.5% VS (100% VS containing 38% (v/v) ethylene glycol (EG), 0.5 m trehalose, and 6% BSA in PBS). The embryos were then loaded into super open pulled straws (SOPS) (Minitub, Tiefenbach, Germany) in a minimum-volume droplet (0.5 μL). The SOPSs containing the embryos were sealed by applying a soldering device to the narrow end and by inserting an identification rod into the wide end. Samples were vitrified at a rapid cooling rate in LN Slush (VitMaster apparatus, IMT Ltd, Ness-Ziona, Israel). On the day of transfer, blastocysts were warmed by plunging the SOPS into the warming chamber of the device, which contained 70% ethanol at 37°C, for 5 s. The straws were then withdrawn from the warming chamber and the sealed end was cut off carefully. The embryos were immersed in a 200-μL drop of 0.6 m trehalose in PBS solution for 4 min and then transferred through a series of solutions containing decreasing concentrations (0.5, 0.4, 0.3, 0.2, and 0.1 m) of trehalose for 2 min each. Viability was evaluated according to the ability of the embryos to re-expand. Blastocysts with the highest morphology rank were selected for transfer and loaded into the transfer gun (Minitub). Holstein recipients were in estrus 6–8 days prior to transfer. In Exp. 1, all blastocysts (n = 15) re-expanded after the vitrification and warming processes and were transferred into the uteruses of 15 Holstein recipient cows. One recipient cow became pregnant and gave birth to a healthy calf. In Exp. 2, all blastocysts (n = 6) re-expanded after warming and were transferred, one each into the uterus of a recipient Holstein heifer. Three pregnancies are still ongoing. In summary, the results obtained demonstrate that plunging embryos in SOPSs into LN Slush in outdoor conditions offers a potential technique for embryo cryopreservation and transfers. Further field trails are required to examine the effect of recipient age (cow vs. heifer) and embryo breed (Charolais vs. Holstein) on the suggested procedure.
12 THE EFFECT OF PROGESTERONE CONTENT IN A VAGINAL INSERT ON PREGNANCY RATES IN BEEF AND DAIRY CATTLE INSEMINATED AT FIXED TIME
Authors: Cutaia L. , Aviles M. , Feresin F. , Franzoni A. , Peres L. , Pincinato D. , Bo G. A.
Published: December 2006

Three experiments were conducted to compare pregnancy rates in cows and heifers treated with intravaginal progesterone (P4)-releasing devices impregnated with either 0.5 or 1.0 g of P4 and inseminated at a fixed time (FTAI). In Experiment 1, 102 postpartum beef cows (Hereford and Angus crossbred with Tuli, 60 to 90 d postpartum) with a body condition score (BCS) of 2.5 to 3.0 (1 to 5 scale) were used. On Day 0, all cows received 2 mg of estradiol benzoate (EB; Syntex, Argentina) i.m. and were randomly assigned to receive an intravaginal progesterone-releasing device with 0.5 g of P4 (DIB 0.5 g; Syntex) or 1.0 g of P4 (DIB 1.0 g; Syntex). On Day 7, DIB devices were removed and all cows received 150 µg D(+)cloprostenol (Ciclase; Syntex) i.m. On Day 8, all cows received 1 mg EB and were FTAI 52 to 56 h after DIB removal. In Experiment 2, 93 Holstein cows that were 90 to 110 days in milk (DIM), producing on average 5000 kg of milk per lactation and with a BCS of 2.5 to 3.0 received the same treatments as those in Experiment 1. In Experiment 3, 100 Holstein heifers, 20 to 22 months old, with a BCS of 2.5 to 3.0, also received the same treatments as those in Experiment 1. Pregnancy rates in all experiments were determined by ultrasonography 35 days after FTAI and compared by chi-square test. No differences in pregnancy rates were detected between lactating beef cows, dairy cows, or dairy heifers treated with DIB devices impregnated with 0.5 or 1.0 g of P4 (Table 1). It was concluded that vaginal P4 releasing devices impregnated with 0.5 g or 1.0 g of P4 results in comparable pregnancy rates in FTAI protocols in beef and dairy cattle.

Table 1.
Pregnancy rates in postpartum beef cows, Holstein cows, and Holstein heifers treated with DIB devices impregnated with 0.5 or 1.0 g of P4 and subjected to FTAI
120 EMBRYO SURVIVAL FOLLOWING LIPID-BASED TRANSFECTION OF 1-CELL STAGE BOVINE EMBRYOS WITH SMALL INTERFERING RNA (siRNA) FRAGMENTS AND/OR DNA
Authors: Bertolini M. , Bertolini L. R. , Petkov S. G. , Madden K. R. , Murray J. D. , Anderson G. B.
Published: December 2006

The RNA interference (RNAi) technology is a powerful tool for studies in functional genomics. The aim of this study was to evaluate the effects of a cationic lipid-based small interfering RNA (siRNA) and/or DNA delivery to 1-cell-stage bovine embryos on survival to the blastocyst stage. In vitro-produced (IVP) embryos were generated according to Bertolini et al. 2002 (Theriogenology 58, 973), and cloned embryos were produced by the handmade cloning technique (Vajta et al. 2003 Biol. Reprod. 68, 571) using green fluorescent protein (GFP)-expressing fibroblast cells as nuclear donors. Lipofections were performed on zona-free 1-cell-stage IVP embryos at 24–28 h post-fertilization by exposure to 1% (v/v) Lipofectamine 2000 (Invitrogen Co., CA, USA), 0.002% (w/v) GFP plasmid (pEFGP-N1, Clontech Laboratories, CA, USA) and/or various doses of siRNA GFP-specific siRNA oligonucleotide (Invitrogen) or DNA methyltransferase 1 (Dnmt1)-specific siRNA fragments for 60 min at 39°C, according to 5 treatment groups: (1) zona-intact IVP embryos (controls), (2) zona-free control embryos (controls for embryo development after zona removal), (3) embryos treated with GFP + GFP-siRNA at 0, 50, 100, 200, 400, or 800 nm, (4) embryos treated with Dnmt1-siRNA at 0, 50, 100, 250, or 500 nm, and (5) cloned embryos (positive controls for GFP expression). After treatment, embryos were in vitro-cultured in a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256) for 7 days. Cleavage and developmental rates to at least 8-cell and to blastocyst stages were assessed at 48, 96, and 168 h post-fertilization (hpf), respectively. Data were analyzed by the chi-square test. Cleavage rates in embryos treated with higher doses of siRNA were lower than in all other groups (Table 1). Embryo survival to at least 8-cell stage at 48 h, based on cleavage, was similar among all treatments (data not shown), but survival to blastocyst stage was affected by higher doses of GFP- or Dnmt1-siRNA (Table 1). After a qualitative assessment by fluorescence microscopy at 168 hpf, 40 to 63% of GFP-transfected blastocysts showed various levels of fluorescence, irrespective of the siRNA treatments. Fragments of siRNA are known to be short-lived in cultured cells, although we are still uncertain of their behavior and effects in early bovine embryos. We are currently analyzing the effectiveness of the siRNA transfection in the early IVP and clone embryo. In conclusion, liposome transfection of 1-cell-stage embryos did not affect survival and development to the blastocyst stage. However, survival followed an siRNA dose-response effect, with doses higher than 400 nm appearing to be detrimental to embryo development, with a developmental arrest at or close to the embryonic genome activation period.

Table 1.
Developmental rate of bovine embryos following lipid-based transfection at the 1-cell-stage
125 ISOLATION OF MULTILINEAGE STEM CELLS FROM THE PORCINE MAMMARY FAT PAD
Authors: Davidson T. , Lane S. , Ferguson C. , Wheeler M. , Hurley W.
Published: December 2006

Porcine mammary tissue is a dynamic system that undergoes multiple cycles of growth, differentiation, and regression during the life cycle of a female; however, the mechanisms of mammary morphogenesis are not fully understood. The current hypothesis suggests that epithelial stem cells in the terminal end buds interact with surrounding epithelial and stromal cells during mammary tissue development and regeneration. Because the mammary gland of the virgin animal consists largely of a pad of adipose tissue, we propose that adipose-derived stem cells from the mammary fat pad (MFP) may also play a role in the regeneration process. Therefore, the objective of this experiment was to determine whether multilineage stem cells are present in the MFP of gilts. Adipose tissue from the MFP and back fat (BF) from the loin region were harvested from six nulliparous, cycling Yorkshire gilts, approximately 16 mo of age. Methods for isolation of adipose-derived stem cells were modified from those of Malusky and Wheeler (2004; International Society for Stem Cell Research, Proceedings 2nd Annual Meeting, abstr. 248:124). Presumptive stem cells isolated from each tissue type were maintained in vitro in DMEM supplemented with either 10% newborn calf serum (NCS; Rep 1 and 2) or 10% fetal calf serum (Rep 3). BF and MFP cell types formed colonies of fibroblast-like cells within 3 to 5 d. Growth characteristics are shown in Table 1. When the cells were 80% confluent, they were trypsinized and reseeded into 75-cm2 flasks. When cultured in the presence of NCS, both cell types had a progressive decrease in viability, and these cultures could not be maintained past Passage 4. Differentiation of both cell types (Passage 1) into adipogenic, myogenic, and osteogenic lineages are currently underway. Based on morphological evaluations, both cell types are able to differentiate into the aforementioned lineages; however, differentiation of MFP-derived cells occurred at a slower rate and was less pronounced than that of BF-derived cells. These preliminary findings suggest that the MFP likely contains a population of multipotent stem cells; however, at this time, it is not possible to make meaningful statistical comparisons. Further experiments are needed to fully characterize these cells and determine their role in mammary gland morphogenesis.

Table 1.
Growth of porcine adipose-derived stem cells obtained from the BF or from the MFP

This study was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program and USDA Multistate Project (W-1171).
127 BINDING RETINOID RECEPTORS BY SPECIFIC AGONISTS AFFECTS THE BOVINE BLASTOCYST DEVELOPMENT IN VITRO
Authors: Gómez E. , Rodríguez A. , Alonso-Montes C. , Caamaño N. , Royo L. J. , Goyache F. , Facal N. , Ikeda S. , Álvarez I. , Díez C.
Published: December 2006

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 �L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM � SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 �M on Day 7 were higher than LG 10 �M, LG 0.1 �M, and LG 0 �M (Day 7: 42.8 � 4.1 vs. 34.4 � 3.7, 36.8 � 3.7, and 32.4 � 3.7, respectively). On Day 8, LG 1 �M also yielded more blastocysts than LG 0.1 �M (50 � 4.2 vs. 44.4 � 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 �M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 �M (42.4 � 2.4 vs. 33.1 � 2.0 and 36.0 � 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 � 13.2 and 96.23 � 13.2, respectively vs. 72.8 � 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 �M blastocysts and expanded controls (39.5 � 5.5 vs. 24.2 � 5.7, and 20.9 � 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts.
This work was supported by grant MCYT, project AGL-2005-04479.