bluuebunny/crossref_metadata_2025 huggingface dataset:
Wireless power transfer based on dielectric resonators with colossal permittivity
Authors: Song Mingzhao , Belov Pavel , Kapitanova Polina
Published: December 2016

Magnetic resonant wireless power transfer system based on dielectric disk resonators made of colossal permittivity (ε = 1000) and low loss (tan δ = 2.5 × 10–4) microwave ceramic is experimentally investigated. The system operates at the magnetic dipole mode excited in the resonators providing maximal power transfer efficiency of 90% at the frequency 232 MHz. By applying an impedance matching technique, the efficiency of 50% is achieved within the separation between the resonators d = 16 cm (3.8 radii of the resonator). The separation, misalignment and rotation dependencies of wireless power transfer efficiency are experimentally studied.
Resonant response in mechanically tunable metasurface based on crossed metallic gratings with controllable crossing angle
Authors: Yachin V. , Ivzhenko L. , Polevoy S. , Tarapov S.
Published: December 2016

We report on a modern class of mechanically tunable planar metamaterials comprising resonating units formed by crossed metallic strip gratings. We observe a resonant response in transmission spectra of a linearly polarized wave passing through a system of crossed gratings in the microwave regime. Each grating consists of an array of parallel metallic strips located on the top of a dielectric substrate. It is revealed that the resonant position appears to be dependent on the angle of crossed gratings. It is found theoretically and experimentally that the resonant shift on the frequency scale appears as a result of increasing in the length of the resonating portion of the parallelogram periodic cell formed by the crossed metallic strips with decreasing crossing angle. The proposed design can be used in innovative types of planar metamaterials and filters.
Significant enhancement of the charging efficiency in the cavities of ferroelectrets through gas exchange during charging
Authors: Qiu Xunlin
Published: December 2016

Ferroelectrets are non-polar polymer foams or polymer systems with internally charged cavities. They are charged through a series of dielectric barrier discharges (DBDs) that are caused by the electrical breakdown of the gas inside the cavities. Thus, the breakdown strength of the gas strongly influences the charging process of ferroelectrets. A gas with a lower breakdown strength has a lower threshold voltage, thus decreasing the onset voltage for DBD charging. However, a lower threshold voltage also leads to a lower value for the remanent polarization, as back discharges that are caused by the electric field of the internally deposited charges can take place already at lower charge levels. On this basis, a charging strategy is proposed where the DBDs start in a gas with a lower breakdown strength (in the present example, helium) and are completed at a higher breakdown strength (e.g., nitrogen or atmospheric air). Thus, the exchange of the gas in the cavities during charging can significantly enhance the charging efficiency, i.e., yield much higher piezoelectric coefficients in ferroelectrets at significantly lower charging voltages.
Flow units perspective on elastic recovery under sharp contact loading in metallic glasses
Authors: Shahzad K. , Gulzar A. , Wang W. H.
Published: December 2016

The obscure nature of glass physics has led to develop various correlations between different parameters and properties of metallic glasses. Despite these correlations, the clear picture of plastic deformation is still lacking. We have measured elastic recovery in metallic glasses by indentation, and found the elastic recovery correlate with different properties and parameters of metallic glasses. All these observations can be quite well explained with flow unit model which could provide clearer picture on the plastic deformations and nature of the metallic glasses.
Shape induced acid responsive heat triggered highly facilitated drug release by cube shaped magnetite nanoparticles
Authors: De Debarati , Mandal Goswami Madhuri
Published: December 2016

This paper reports a very simple yet better method for synthesis of cube shaped magnetite nanoparticles (MNPs) and their application in the drug delivery system (DDS). Structural analysis was done by XRD measurements to confirm the phase of the material, and morphological information was obtained through TEM analysis to confirm the shape and size of the particles. It has been shown that these particles can be decomposed in acid medium. These acid-decomposable magnetite nano-particles have been used for heat triggered, remote-controlled, on demand delivery and release of a cancer drug doxorubicin for research and therapeutic purposes. Here, we have shown that the pH stimulated and heat-triggered release of drug from our MNPs significantly enhances the release efficiency. In this case, we observe that pH induced release is far better in comparison to heat-triggered release. From these inspiring results, it may be expected that this methodology may become a significant step towards the development of a pH-sensitive heat triggered drug delivery system minimizing drug toxicity.
101 DOSE AND TIMING OF ADMINISTRATION OF PROSTAGLANDIN F2α DURING FIXED-TIME EMBRYO TRANSFER IN AN IN VITRO-PRODUCTION PROGRAM
Authors: Sala R. V. , Garcia-Guerra A. , Carrenho-Sala L. C. , Fosado M. , Peralta E. , Tosta R. D. , Tosta L. C. C. , Moreno J. F. , Baruselli P. S. , Wiltbank M. C.
Published: December 2016

Synchronization protocols for fixed-time embryo transfer (ET) contribute significantly to the overall cost of an in vitro-produced-ET program, primarily through the cost of drugs and the labour required. Optimization of synchronization protocols to reduce cost, while providing high fertility, have the potential to improve overall efficiency and profitability. The objective of the present study was to evaluate the effect of dose and schedule of administration of prostaglandin F2α (PGF) during a synchronization protocol for fixed-time ET. Holstein and cross-bred Holstein heifers (n = 3766) were synchronized using a modified 5-day CIDR Synch as follows: Day 0: CIDR inserted; Day 5: CIDR removed, PGF2α treatment; Day 8: gonadotropin-releasing hormone (GnRH; 100 μg of gonadorelin). On Day 5, at the time of CIDR removal, heifers were randomly assigned to a 2 × 2 factorial design to receive either a full or half dose of PGF (Cloprostenol; 500 v. 250 μg) and 1 (Day 5) or 2 (Day 6) administrations resulting in the following treatments: full dose Day 5 + Day 6 (n = 938); full dose Day 5 (n = 938); half dose Day 5 + Day 6 (n = 946); and half dose Day 5 (n = 944). Heifers were evaluated by ultrasonography 5 days after GnRH to determine presence and size of the corpus luteum. Heifers with a corpus luteum received a fresh in vitro-produced embryo 7 ± 1 days after GnRH administration, and pregnancy was determined by ultrasonography 32 and 60 days after GnRH. Fertility data were analysed by logistic regression and included the fixed effects of dose, time, and their interaction. Fertility results are shown in Table 1. Utilisation rate (transferred/treated) was not affected by dose (P = 0.66), time (P = 0.19), or their interaction (P = 0.17). The percentage of heifers detected in oestrus was not affected by dose (P = 0.13), time (P = 0.72), or their interaction (P = 0.89). There were no significant differences between doses of PGF (P = 0.32), time (P = 0.71), or their interaction (P = 0.80) on pregnancies per ET on Day 32. Similarly, no differences were found on pregnancies per ET on Day 60 between doses (P = 0.35), time (P = 0.96), or their interaction (P = 0.89). In addition, pregnancy loss between Day 32 and 60 was not affected by dose (P = 0.76), time (P = 0.66), or their interaction (P = 0.54). In conclusion, the use of a half dose of PGF once on Day 5 resulted in comparable utilisation rate and fertility as the observed with 2 full dose applications 24 h apart. As a result, the overall cost of the fixed-time ET program can be reduced by eliminating the need for a second PGF treatment and by decreasing the dose without compromising fertility.

Table 1.
Utilisation rate, oestrus expression, pregnancies per ET (P/ET), and pregnancy loss in recipients receiving either a full or half dose of prostaglandin F2α on Days (D) 5 and 6 or once on D 5
126 EXPRESSION OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS IN BOVINE OOCYTES AND EMBRYOS CULTURED IN LOW AND HIGH OXYGEN
Authors: Barandalla M. , Colleoni S. , Galli C. , Lazzari G.
Published: December 2016

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that binds a broad repertoire of advanced glycation end products ligands. It is constitutively expressed at a high level during embryonic development, but its levels decrease in adult tissues. The RAGE-ligand interaction induces a series of signal transduction cascades and leads to the activation of several inflammatory signalling pathways. This is why RAGE is frequently associated with pro-inflammatory responses and it is implicated as an underlying condition in immune disorders, cardiovascular diseases, diabetes, Alzheimer’s disease, and cancer. However, the physiological function of RAGE during embryogenesis and in aging is largely undefined. The aim of this work was to determine the basal levels on RAGE expression in bovine oocytes (germinal vesicle, GV, and in vitro-matured metaphase II) and in vitro-derived embryos developed in SOF medium, by qPCR (3 replicates each containing pooled RNA from 20 oocytes or embryos, t-test P < 0.05) and immunostaining. The expression of RAGE was similar in bovine oocytes, in cleaving embryos up to morula, and afterward declined at the blastocyst stage. However, a more detailed investigation, by the separation of the inner cell mass (ICM) from the trophoblast, indicated that expression decreased only in the trophoblast (over 5-fold) while it was maintained high in the isolated ICM. In the trophoblast, compared to the ICM, this finding coincided with an elevated expression of PSMA6 gene, which encodes for the 20S proteasome subunit, and ubiquitin (UBB) gene, suggesting a link between proteasome activity and RAGE expression. Then we investigated the expression of RAGE and the proteasome-ubiquitin system in embryos cultured under different oxygen concentrations [i.e. physiological (5%) or high (20%) O2 levels]. Preliminary results showed that RAGE expression was lower in embryos cultured in 20% O2 and conversely the expression of PSMA6 and UBB genes was higher. Thus, these results indicate that the activity of RAGE-mediated stress response pathway in pre-implantation bovine embryos is affected by O2 tension during in vitro culture.
This work was funded by projects Epihealth FP7 n. 278418, EpiHealthNet FP7-PEOPLE-2012-ITN n. 317146, and Fecund FP7 n. 312097.
134 RELAXIN AND ITS RECEPTORS IN MATURE CANINE SPERMATOZOA
Authors: Almeida I. L. G. , Durfey C. L. , Gastal G. D. A. , Devos-Burnett D. , Willard S. T. , Ryan P. L. , Feugang J. M.
Published: December 2016

Relaxin and its receptors (RXPF1 and RXFP2) are found in reproductive and non-reproductive tissues of both males and females, across species. In dogs, both relaxin and receptors are found in reproductive tissues of females and play important roles during pregnancy; however, their presence in male reproductive tissues remains unclear. The goal of this study was to explore the presence of both relaxin and receptors in mature dog spermatozoa. Semen samples of 4 adult (≥4 years old) dogs were harvested from a local breeder and diluted (vol:vol) with a pre-warmed Tris extender solution (pH 6.9). Samples were centrifuged and sperm pellets were resuspended in the Tris extender for motility and velocity analyses using computer-assisted sperm analyzer. Semen with over 70% motility were retained for analyses. A subset of those samples (n = 4 dogs) was subjected to total RNA extraction, followed by RT-PCR to amplify relaxin and RXFP1/2 RNA transcripts, with β-actin used as the housekeeping gene. All PCR amplicons were run on a gel electrophoresis for imaging. Another subset of samples (n = 4 dogs) were used for total protein extraction, followed by immunoblotting using anti-human RXFP1 and RXFP2 antibodies. Finally, the remaining subset of samples (n = 4 dogs) was fixed in 4% paraformaldehyde and submitted to a standard procedure of immunofluorescence using an anti-human relaxin, and followed by fluorescein isothiocyanate-conjugated secondary antibody. Labelled spermatozoa were immediately analysed with flow cytometry and imaged with a confocal microscope. Gene expression data were collected after 40 cycles of PCR and all samples expressed β-actin. The detection of RXFP2 was observed at a low level in all samples, whereas RXFP1 and relaxin were absent. Protein analysis with western immunoblotting showed weak signal bands of both receptors in all samples; however, the band corresponding to RXFP1 (~82 kDa) appeared stronger than that of RXFP2 (~90 kDa). Flow cytometry revealed small proportions of relaxin immunopositive spermatozoa (less than 35%) that exhibited low fluorescence intensity. Confocal microcopy imaging confirmed the weak signals that were mostly located in the sperm head. Altogether, the findings showed that relaxin and its receptors RXFP1 and RXFP2 are present in mature spermatozoa of adult dogs. More studies are needed to better characterise the roles of the relaxin system on canine sperm fertility.
Grant supports are from the USDA-ARS Biophotonics Initiative project #58–6402–3-018 and Brazil Scientific Mobility Program (BSMP) scholarship from CAPES-Brazil.
143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION
Authors: Yamanouchi T. , Sugimura S. , Matsuda H. , Ohtake M. , Goto Y. , Kobayshi S. , Hashiyada Y.
Published: December 2016

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.
159 IN VITRO DEVELOPMENT OF CAPRINE EMBRYO USING CRYOPRESERVED BLACK BENGAL BUCK SEMEN
Authors: Kumar R. , Chandra P. , Konyak P. , Karunakaran M. , Santra A. , Das S. K.
Published: December 2016

The purpose of this study was to check the competence of cryopreserved black Bengal buck semen to produce goat embryo through IVF. So far, cryopreserved black Bengal buck semen has not been used to produce goat embryos by IVF. For the study, fresh goat oviducts and ovaries were collected from slaughterhouse in a thermo flask containing 0.9% saline solution supplemented with antibiotic (400 IU mL−1 penicillin and 500 mg mL−1 streptomycin) at 30–35°C and transported to laboratory within 2–3 h of slaughter. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, washed 5–6 times, and cultured in maturation media (TCM-199 + 10% FBS + 10 mg mL−1 FSH-P + 0.81 mM sodium pyruvate + 5% follicular fluid + 50 mg mL−1 gentamicin sulfate + 1 μg mL−1 oestradiol + 100 μM cysteamine) for 27 h in a 5% CO2 incubator at 38.5°C with maximum humidity. After 27 h of culture, cumulus cells were separated from matured oocytes by repeated pipetting using a fine pipette in fertilization Bracket and Oliphant’s (BO) medium. After removal of cumulus cells, the oocytes were transferred to acidified Tyrode’s medium for zona thinning for 52 s and were co-incubated with capacitated sperms for fertilization in fertilization BO medium at 38.5°C in 5% CO2 in air with maximum humidity. In the experiment I, freshly collected buck semen was used for IVF after processing for capacitation. In experiment II, cryopreserved buck semen straws were thawed and sperm were capacitated in vitro and used for fertilization. After 5 h of co-incubation, presumptive zygotes were washed thoroughly and cultured in embryo development medium for cleavage. Three different in vitro development media (RVCL, mSOF + 2.5% BSA, and KSOM + 0.5% BSA) were used. After 40 to 42 h, cleavage was observed and embryos were co-incubated with oviducal cells in replacement media for further development. In the fresh group, overall cleavage rates (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74 and morula formation rates (%) were 7.72 ± 3.38, 6.03 ± 1.29, 3.00 ± 3.00 in RVCL, mSOF, and KSOM media, respectively. However, in the cryopreserved group the overall cleavage rates (%) were 29.17 ± 2.56, 27.70 ± 2.31, and 24.17 ± 1.44 in RVCL, mSOF, and KSOM media, respectively, and morula formation (%) was achieved 2.93 ± 0.97 in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development through IVF.
16 COMPARATIVE STUDY ON EPIDIDYMAL SPERM TRAITS OF BONSMARA AND NGUNI BULLS
Authors: Maqhashu A. , Netshirovha T. R. , Bovula N. , Makhanya L. G. , Sebei P. J. , Matabane M. B. , Mphaphathi M. L. , Nedambale T. L.
Published: December 2016

The recovery and preservation of sperm from the epididymides of slaughtered breeding bulls is a viable option in maintaining their germplasm for artificial insemination or for future use in breeding programs. The aim of this study was to evaluate and compare the quality of epididymal sperm derived from slaughtered Bonsmara and Nguni bulls. The study used 20 bulls that were 2 years of age, 10 from each breed. Twenty pairs of testes from Bonsmara and Nguni bulls were removed by orchiectomy within 20 min of slaughter, and semen was extracted from the cauda epididymis. The testicle length and weight were measured. Semen from bulls of the same breed was pooled and sperm characteristics were evaluated using a computer-aided sperm analysis (CASA). Collected semen samples were then extended (1:1) in egg yolk citrate (EYC) extender and stored at 5°C. Sperm motility rate was evaluated at 0, 24, 30, and 48 h during in vitro semen storage. Data were analysed by ANOVA. The Bonsmara bull’s testicle length (13 ± 0.31cm) and weight (290 ± 0.24 g) were higher than that of the Nguni bulls (10.3 ± 0.11 cm and 275 ± 0.42 g; P < 0.05). However, there were no significant differences in fresh total sperm motility rate (74.4 ± 2.14 v. 75.8 ± 3.11%) or concentration (5.31 ± 1.14 v. 5.83 ± 2.09 × 109) of Bonsmara and Nguni bulls, respectively. There were no significant differences observed for total sperm motility at 24 h (49.3 ± 1.18 v. 52.6 ± 2.60%) and 30 h (40.5 ± 1.94 v. 43.4 ± 2.81%) between the Bonsmara and Nguni bulls, respectively. The total sperm motility at 40 h was <20% for both Bonsmara and Nguni bulls on the EYC extender. There were no differences in sperm motility rate of the epididymal semen of Bonsmara and Nguni bulls. We concluded that it is possible to derive semen from the epididymis of indigenous Nguni and composite Bonsmara bulls.
160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS
Authors: Negota N. C. , Nethenzheni L. P. , Mphaphathi M. L. , Barry D. M. , Nedambale T. L.
Published: December 2016

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.
161 EFFECTS OF LIPOSOMES ON SPERM MOTILITY AND DNA-BINDING EFFICIENCY
Authors: Lotti S. N. , Rubessa M. , Knox R. V. , Wheeler M. B.
Published: December 2016

In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi et al. 1989 J. Reprod. Fert. Abstr. Ser. 4) and cattle (Perez et al. 1991 Biotecnol. Apl. 8, 90–94). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analysed methods to select thawed bovine sperm for DNA binding and evaluated the binding of exogenous DNA to those sperm. To determine the optimal sperm-selection method, the sperm were analysed using a computer-assisted sperm analyzer (CASA), the parameters selected were: total motility, rapid motility, and progressive motility. To measure the binding of DNA we used an indirect analysis using NanoDrop technology (Thermo Scientific, Wilmington, DE, USA) to compare the different DNA concentrations among groups. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate and a neutral lipid, l-a Dioleoyl phosphatidyl-ethanolamine prepared according to the protocol of Russell (1997). Percoll or swim-up methods were used to select sperm after thawing (Rubessa et al. 2016), followed by incubation (3 h) with the liposome-DNA complexes according to liposome preparation protocol (Russell, 1997). We used enhanced green fluorescent protein in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analysed: 1) immediately after processing (Control), 2) After 3 h of incubation with no liposomes, 3) incubation with liposomes and no DNA, 4) incubation with 1 ng of DNA, and 5) incubation with 10 ng of DNA. This was repeated five times. The CASA results for total motility and rapid motility showed a greater amount of significant differences (P < 0.01) between the control and the other treatments in the Percol group as opposed to swim-up. These results confirm that the sperm selected with swim-up is more stable. Following CASA analysis, sperm was washed with PBS twice and collected in tubes. The DNA from all samples was extracted to determine the quantity of attaching varying amounts of DNA to sperm. The results showed a general increase in DNA concentrations with the increase of DNA added for both methods, but the statistical variation was too large to draw any definite conclusion. In future studies, real-time PCR will be used to determine the quantity of enhanced green fluorescent protein bound to the sperm.

Table 1.
Results of the computer-assisted sperm analyzer (CASA)
185 OVIDUCTAL CO-CULTURE CELL DID NOT REDUCE THE RATE OF CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Demyda-Peyrás S. , Peral-García P. , Moreno-Millán M.
Published: December 2016

One of the major causes of embryo failure on in vitro-produced cattle embryos is their increased rate of chromosomal abnormalities. It was demonstrated that they can be increased by the pre- and post-environmental conditions of culture, such as the culture media and supplementation and culture atmosphere among others. Furthermore, it was suggested that the percentage of chromosomal abnormalities detected could be used as an indirect methodology to evaluate the embryo stress during culture. The use of oviduct cells in co-culture was employed as a technique that improves the embryo quality in different culture systems in cattle, probably by modulating the variations in the embryo environment or detoxifying the culture media. For that reason, we aimed to determine if the use of an oviducal co-culture system could reduce the percentage of chromosomal abnormalities in in vitro-produced cattle embryos. Oviducal cells were obtained from abattoir oviducts, and plated and cultured in TCM-199 media following standard procedures. Cumulus-oocyte complexes were also obtained from the abattoir and matured in a standard media (TCM199, with glutamine, pyruvate, FSH, LH, and antibiotics) for 20 h (38.5°C, 5% CO2). Matured oocytes were fertilized in IVF-TALP with 1 × 106 spermatozoa/mL (previously selected through a 45–90% Percoll™ gradient) for 18 h using the same conditions. Presumptive zygotes (n = 653) were divided into 2 groups and cultured in the mSof medium for 72 h, with (SOV) or without (SOF) the addition of oviducal cells. In total, 108 embryos were successfully karyotyped following our standard laboratory techniques. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a bright-field microscope using a simple Giemsa staining. The percentage of diploid embryos (54/58 in SOV and 47/53 in SOF) and abnormal embryos (4/58 in SOV and 6/53 in SOF) were nonsignificant (P > 0.05; Z-score test for 2 population proportions). Our results suggest that the use of oviducal cells as a co-culture supplementation in in vitro-produced cattle embryos do not improve the percentage of chromosomal abnormalities detected.
23 THE EFFECT OF WITHDRAWAL TIMING OF CONTROLLED INTERNAL DRUG RELEASE (CIDR) ON EWE REPRODUCTIVE PERFORMANCE
Authors: Swelum A. , Moumen A. , Alowaimer A.
Published: December 2016

This study was carried out using 80 multiparous Awassi ewes during breeding season to compare the effects of controlled internal drug release (CIDR) withdrawal time on ewe fertility. Ewes were equally and randomly allotted into 4 groups (n = 20/group). Ewes had a CIDR inserted for 3, 6, 9, or 12 days with intramuscular administration of 300 IU of equine chorionic gonadotropin (eCG) at withdrawal time. Oestrus was detected using vasectomized ram starting 12 h after CIDR withdrawal and repeated every 12 h up to 84 h. Blood samples were collected from all groups at the time of CIDR withdrawal for measuring of oestradiol (E2) and progesterone (P4) serum concentrations using commercial ELISA kits and micro-titrimetric plates. Timed insemination was performed 48 h post CIDR withdrawal in all groups. Pregnancy was diagnosed by ultrasonography at day 23 post-insemination and confirmed at day 35. Comparisons among groups were evaluated using Chi Square (χ2) test in all measured parameters except hormones levels, which analysed by repeated-measures analysis of variance (ANOVA), using SAS (SAS Institute Inc., Cary, NC, USA). A difference was considered significant at P < 0.05 level. The results revealed that the retention, vaginal discharge and drawstring breakage rates after CIDR removal were insignificantly differed between all groups. On the other hand, pregnancy rate was significantly (P ≤ 0.05) higher in 9-d and 6-d groups (68.4 and 60%, respectively) than the 3-d group. While, no significant difference was detected between 12d group and other groups. The heat detection rate was significantly (P ≤ 0.05) higher in 12d group (100%) than 6-d and 3-d groups (80 and 45%, respectively). While, no significant difference was detected between 12-d and 9-d groups in heat detection rate. P4 was significantly higher in the 12-d group (13.4 ± 3.06 ng mL−1) than other groups. While E2 was significantly lower in the 12-d group (1.6 ± 0.06 pg mL−1) than other groups. These results indicated that withdrawal of CIDR devices after 9 days are efficient in synchronizing oestrus in ewes and provided higher pregnancy rate.
28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO
Authors: Kim Y. J. , Ahn K. S. , Park S. M. , Lee B. C. , Shim H. , Ahn C.
Published: December 2016

For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.
4 SUBFERTILITY IN BULLS CARRYING A NONSENSE MUTATION IN TMEM95 IS DUE TO FAILURE TO PENETRATE THE ZONA PELLUCIDA
Authors: Fernandez-Fuertes B. , Kölle S. , Lonergan P.
Published: December 2016

In a recent genome-wide association study, male reproductive ability was assessed for 7962 Fleckvieh sires (Pausch et al. 2014 PLoS Genet. 10, e1004044). Forty bulls in the population with exceptionally poor reproductive performance were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95)-encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, out of 35,671 inseminations with these bulls, only 1.7% resulted in pregnancies. In addition, none of the bulls with normal reproductive performance was homozygous for this mutation. The aim of this study was to examine the effect of this nonsense mutation in TMEM95 on bovine sperm function in vitro. In all experiments, data were assessed for normality of distribution and analysed using a one-way ANOVA. The model included the main effects of treatment, replicate and their interactions. In Experiment 1, the fertilizing ability of sperm, assessed as oocyte cleavage rate, from 5 homozygous (mt/mt), 3 heterozygous (wt/mt), and 2 wildtype (wt/wt) Fleckvieh bulls was assessed. Oocytes fertilised with sperm from mt/mt males had a lower cleavage rate at 48 h post-fertilization than oocytes fertilised with sperm from wt/wt or wt/mt bulls (26 ± 3%, 72 ± 2%, and 79 ± 3%, respectively; P < 0.01). In addition, the kinetics of cleavage were slower in the mt/mt group as evidenced by a lower proportion of embryos beyond the 2-cell stage at 48 h (32 ± 12.6%, 90 ± 3.6%, and 87 ± 0.4%, respectively, P < 0.01). This translated into a lower blastocyst rate at Day 8 in the mt/mt group in comparison with wt/mt and wt/wt groups (2 ± 1%, 27 ± 3%, and 40 ± 2%, respectively, P < 0.01). In Experiment 2, fluorescent staining of oocytes 3 h after fertilization revealed a lower number of sperm from mt/mt bulls bound to the zona pellucida (ZP) in comparison with sperm from wt/wt or wt/mt individuals (3 ± 1.0, 11 ± 2.3, and 24 ± 9.4, respectively; P < 0.05). In order to further study the potential role of TMEM95 in sperm-ZP interaction, in Experiment 3 sperm from the 3 groups were used to fertilize ZP-free oocytes. Consistent with the previous results, sperm from mt/mt bulls were less able to penetrate oocytes with no ZP than sperm from wt/wt or wt/mt bulls (6 ± 2%, 44 ± 8%, and 68 ± 14%, respectively; P < 0.05). In conclusion, these results suggest that TMEM95 is involved in events that take place before ZP binding but are required for sperm to interact normally with the oocyte vestments and, ultimately, to achieve fertilization. Further study of the possible role of this protein in the initiation of sperm capacitation and hypermotility, as well as in early embryo development, will shed more light regarding the crucial role of TMEM95 in sperm function.
Supported by the Irish Department of Agriculture, Food and The Marine under the Research Stimulus Programme (Grant No. 11 S 104).
42 THE EFFECT OF VARIOUS CRYOPROTECTIVE AGENTS AND SLOW COOLING RATE ON VIABILITY OF SHEEP OVARIAN TISSUE
Authors: Seisenbayeva A. S. , Toishibekov Y. M. , Iglmanov U. I. , Valieva B. A.
Published: December 2016

Priority objects of protection in agrobiocenoses are grades of cultural plants and local breeds of the cultivated animals. The most general criteria for preservation of local breeds are viability, adaptability, a state of health, reproductive abilities, and unique genetic polymorphism at the molecular and morphological levels. Therefore, the aim of this study was to compare the effectiveness of different cryoprotectors on morphology of ovine ovarian tissue cryopreserved by a passive cooling method. Ovarian tissue from 10 indigenous Chuyi breed was transported to the laboratory within 30 min at 32 to 34°C, divided into smaller pieces (2.0 × 1.2 × 1.0 mm), and randomly distributed into 4 groups: (1) control (fresh tissue), (2) pieces after freezing/thawing with 1.5 M ethylene glycol (EG); (3) pieces after freezing/thawing with 1.5 M propanediol (PROH); (4) pieces after freezing/thawing with 1.5 M dimethyl sulfoxide (DMSO). The ovarian pieces were placed in a cryovial and equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M cryoprotectors with 0.5 M sucrose (5 min each), precooled at 4°C, and stored in a −80°C freezer for 24 h. Then, the cryovials containing the ovarian pieces were placed in liquid nitrogen and stored (for 2 months) until thawing. The ovarian pieces were cultured in vitro for 7 days in TCM-HEPES+ 10% native ovine serum (NOS) (Seisenbayeva et al. 2015 Reprod. Fertil. Dev. 28, 193–194). After 7 days of culture, we evaluated the effects of passive cooling methods with different cryoprotectors on ovarian tissue morphology by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged follicles was counted. All morphologically normal primordial, primary, and secondary follicles had healthy and intact oocytes, each containing a round nucleus and clearly visible nucleolus surrounded by well-organised granulosa cells without a pyknotic nucleus. Integrity rate of tissue after treatment was evaluated by Student’s test. In groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm3 was 17.0 ± 4.6a, 16.2 ± 7.2b, 13.0 ± 5.1c, and 11.9 ± 4.8d, respectively (ab, ac, ad P > 0.05). For these groups, respectively, 69.2 ± 8.2%a , 61.7 ± 8.6%b , 52.4 ± 8.8%c and 48.5 ± 8.8%d preantral follicles were morphologically normal (ab, ac, ad P > 0.05). We did not find significant differences between groups. The analysis of comparative histology shows that 1.5 M EG is more effective on viability of ovarian follicles than 1.5 M DMSO or 1.5 M PROH.
47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)
Authors: Fujikawa T. , Kubota C. , Ando T. , Imamura S. , Tokumaru M. , Yamakuchi H. , Gen Y. , Hyon S.-H.
Published: December 2016

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.
5 TESTICULAR GnRH-II RECEPTOR KNOCKDOWN IMPAIRS DIURNAL TESTOSTERONE SECRETION IN THE BOAR
Authors: Desaulniers A. T. , Cederberg R. A. , Lents C. A. , White B. R.
Published: December 2016

The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are ubiquitously expressed, with elevated levels in the testis. Gene coding errors prevent their production in many species, but both genes are functional in swine. We demonstrated that GnRHR-II localizes to porcine Leydig cells, and exogenous GnRH-II robustly stimulates testosterone production in vivo, despite minimal luteinizing hormone (LH) secretion. These data suggest that GnRH-II directly effects steroidogenesis in the boar testis. To explore this hypothesis, we produced a GnRHR-II knockdown (KD) swine line. Upon characterisation of this line, serum testosterone concentrations were reduced in GnRHR-II KD compared with littermate control males during pubertal development. However, concentrations of LH were unaffected, indicating that GnRHR-II KD impairs steroidogenesis directly at the testis rather than inhibiting gonadotropin secretion from the anterior pituitary gland. Based on these results, the objective of this study was to compare diurnal secretory patterns of testosterone in mature GnRHR-II KD (n = 5) and littermate control (n = 5) males. Boars were fit with indwelling jugular cannulae and blood was collected every 15 min for 8 h. Serum was assayed for testosterone concentration via radioimmunoassay. Next, GnRHR-II KD (n = 5) and littermate control (n = 4) boars were killed, testis weight was recorded, and testicular tissue was collected for RNA isolation. To confirm KD in these animals, digital droplet PCR was performed to quantify GnRHR-II mRNA abundance (normalized to β-actin). Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with line (transgenic or control) as the fixed effect and litter as a random effect. For hormone data, time and line × time interaction were included as fixed effects, with time as a repeated measure. Although there was no effect of time or line × time interaction (P > 0.05) on serum testosterone concentrations, we observed a line effect (P < 0.05). Differences between lines were dramatic; testosterone was reduced by 82% in GnRHR-II KD (0.75 ± 0.05 ng mL−1) compared with littermate control (4.09 ± 0.29 ng mL−1; P < 0.05) males. Despite divergent testosterone levels, testis weights were similar between lines (P > 0.05) indicative of altered Leydig cell function as opposed to hypertrophy/hyperplasia. Given that testicular GnRHR-II mRNA levels were reduced by 69% in transgenic animals (P < 0.001), these data demonstrate that GnRH-II and its receptor play a critical role in testosterone biosynthesis within porcine Leydig cells. Thus, this report reveals novel mediators of testicular function in the boar and challenges the central dogma of testosterone regulation. Because testosterone dictates male reproductive success, GnRH-II and its receptor represent unique targets to improve fertility in swine.
This study was partially supported by NIFA Hatch (NEB-26-199; BRW) and AFRI (2011-67015; CAL) funds.