bluuebunny/crossref_metadata_2025 huggingface dataset:
3 ROLE OF β-DEFENSIN 126 IN PROMOTING SPERM MOTILITY IN CATTLE
Authors: Fernandez-Fuertes B. , Narciandi F. , Meade K. G. , O'Farrelly C. , Fair S. , Lonergan P.
Published: December 2015

As immature sperm migrate through the epididymis, they are bathed in region-specific epididymal fluid, which leads to a sequential addition, deletion, and modification of their surface proteins. These changes ultimately result in the acquisition of motility and fertilising abilities. Among the hundreds of proteins secreted by the epididymis, several β-defensins have been identified and correlated with male fertility in multiple species. In cattle, β-defensin 126 (BD126) is exclusively detected in the reproductive tract of pubertal males, with preferential mRNA expression in the epididymis. Both the macaque and human orthologs have been shown to play a role in the ability of sperm to migrate through cervical mucus. The aim of this study was to examine the role of bovine BD126 in sperm function. Western blot revealed that the peptide is uniquely present in both the cauda epididymis sperm and fluid and is absent from sperm recovered from other proximal epididymal regions, or the ejaculate of vasectomised animals. Confocal analysis showed immunofluorescent labelling of BD126 specific to the tail and acrosomal region in cauda sperm only, suggesting a role in motility. We hypothesised that addition of cauda epididymal fluid (CEF) or recombinant BD126 (rBD126) to immature corpus sperm would improve ability to penetrate cervical mucus. Testes from adult bulls were collected at an abattoir, and sperm from the corpus and cauda epididymis, as well as CEF, were recovered. Corpus sperm were incubated for 1 h with CEF in the absence or presence of BD126 antibody, or with different rBD126 concentrations (30 or 60 μg mL–1); untreated corpus and cauda sperm were used as controls. A higher number of cauda than corpus sperm migrated through cervical mucus from oestrus cows (P < 0.001), and addition of CEF increased the number of corpus sperm migrating through this matrix (P < 0.05). The presence of the BD126 antibody in CEF failed to abrogate this effect. Western blot analysis of the sperm samples revealed the antibody was not successful in blocking BD126 from binding onto the sperm surface, which would explain the lack of differences observed. Furthermore, the addition of rBD126 did not increase corpus sperm migration through mucus. In conclusion, we have characterised the expression of bovine BD126 protein in the bovine testis and epididymis. Incubation of sperm from the corpus with CEF from the cauda resulted in enhanced sperm migration through cervical mucus. However, incubation of sperm with rBD126 in the absence of other factors and proteins from the CEF failed to produce the same effect. These results suggest that the role of BD126 in cattle is different from that observed in primates. We are currently investigating other roles of BD126 and related β-defensins in mediating bovine sperm function.
This work was supported by a grant from the Irish Department of Agriculture, Food and The Marine under the Research Stimulus Programme (Grant No. 11S 104).
36 EFFECT OF cAMP MODULATORS DURING OOCYTE IN VITRO MATURATION ON GAP JUNCTIONAL ACTIVITY OF VITRIFIED BOVINE OOCYTES
Authors: Monteiro C. A. S. , Leal G. R. , Saraiva H. F. R. A. , Camargo A. J. R. , Rosa P. M. S. , Rodrigues A. L. R. , Serapião R. V. , Nogueira L. A. G. , Camargo L. S. A. , Garcia J. M. , Oliveira C. S.
Published: December 2015

Oocyte cryopreservation is a strategic tool for in vitro embryo production, but low rates of cryosurvival are reported for bovine oocytes. Simulated physiological oocyte maturation system (Albuz et al. 2010 Hum. Reprod. 25, 12) uses cAMP modulators to increase oocyte competence by the extension of meiosis block and gap junctional communications activity. The aim of this study was to investigate the effect of simulated physiological oocyte maturation system on gap junctional activity of vitrified bovine oocytes. Oocytes from slaughterhouse ovaries were divided into 4 groups: C (control: fresh immature oocytes); V (vitrified immature oocytes); PM-V (vitrified oocytes after a 2-h pre-in vitro maturation phase – in the presence of AMPc modulators, 100 μM Forskolin, and 500 μM IBMX); and PM (fresh immature oocytes subjected to pre-in vitro maturation). Viable oocytes (n = 404 obtained from 4 replicates) were stained with Calcein-AM using the protocol of Thomas et al. (2004 Biol. Reprod. 71(4), 1142–1149) in order to measure gap junctions activity. Images were captured in fluorescence microscope, and fluorescence intensity was analysed with ImageJ software. Mean fluorescence intensity of each group was normalized to control group to obtain relative intensity value. Means were compared by Kruskal-Wallis test and Dunn post-test. A second analysis was performed considering the percentage of each staining pattern (low, middle, and high intensity) for each group. Results were analysed using Fisher exact test. All statistical analysis were performed in GraphPad Instat program with 5% significance level. Results demonstrated that all treatments induced an increase (P < 0.05) in fluorescence intensity (V: 1.76 ± 1.13; PM-V: 1.58 ± 0.98; PM: 1.38 ± 0.94) compared with control (C: 1.00 ± 0.48). Regarding the staining patterns analyses, immature vitrified oocytes (V group) differed from control group in middle and low patterns (G1, calibrator – high: 11.2%ab, middle: 43.8%a, low: 44.9%a; G2 – high: 8.2%ab, middle: 63.9%b, low: 27.9%b; G3 – high: 16.3%a, middle: 42.3%a, low: 41.3%a; G4 – high: 6.7%b, middle: 53.9%ab, low: 39.3a). In conclusion, unexpectedly, vitrification also increased gap junctional activity, as was found for pre-in vitro maturation group. However, staining pattern analysis results showed only vitrified group was different from control, suggesting vitrified and pre-in vitro maturation groups could have gap activity affected by different ways.
This research was supported by FAPERJ (E26/111.61/2013) and CAPES.
41 DEVELOPMENTAL POTENTIAL OF BUFFALO OOCYTES VITRIFIED AT THE GERMINAL VESICLE STAGE: EFFECTS OF DIFFERENT CRYOPROTECTANT COMBINATIONS AND CRYODEVICES
Authors: El-Shalofy A. S. , Moawad A. R. , Darwish G. M. , Ismail S. T. , Badawy A. B.
Published: December 2015

The cryopreservation of immature oocytes would generate a readily available, nonseasonal source of female gametes for both research and reproduction. In domestic animals, the most promising results in the field of oocyte cryopreservation have been reported in cattle, and a few experiments have been conducted on buffalo. The aim of the present study was to compare the effects of different cryoprotectant combinations and different cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle stage. Cumulus-oocyte complexes obtained at slaughter from mature buffalos were vitrified by using either straw or open pulled-straw or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol or 20% EG + 20% dimethylsulfoxide (DMSO). Following vitrification and warming, viable oocytes were matured in vitro for 22 h. Matured oocytes were either evaluated for nuclear maturation or fertilized and cultured in vitro for 7 days. Recovery rate was significantly higher (P < 0.05) in the oocytes vitrified by straw in 20% EG + 20% glycerol (92.6%) as compared with the other groups. Percentages of viable oocytes were significantly higher (P < 0.05) in the oocytes vitrified in 20% EG + 20% DMSO using SSV (95.7%) than those in the other groups (from 80 to 88.0%). Among the vitrified groups, the highest maturation rate was achieved in SSV with 20% EG + 20% DMSO group (56.7%). This value was comparable with those in the control group (62.1%). After IVF and embryo culture, the highest cleavage and blastocyst rates were obtained in SSV with 20% EG + 20% DMSO group (35.7 and 21.4%, respectively), and these values were nearly similar to those in the control group (38.7 and 25.8%, respectively). Vitrification of oocytes by straw or open pulled-straw resulted in significantly lower (P < 0.05) blastocyst rates (2.6 and 11.5%) as compared with the control. In conclusion, buffalo oocytes vitrified at the germinal vesicle stage can be matured, fertilized, and developed in vitro and produce high frequencies of blastocyst embryos. Solid surface vitrification may be superior to straw and open pulled-straw in vitrification of immature buffalo oocytes because this technique results in higher survival and embryo development rates.
54 IMMUNOLOCALIZATION OF STEROID SULFATASE AND ESTROGEN-SPECIFIC SULFOTRANSFERASE IN BOVINE FOLLICLES
Authors: Blaschka C. , Schuler G. , Wrenzycki C.
Published: December 2015

Steroid hormones are regulators in the fine-tuned mechanism of follicular development in cattle. Their concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes them from being hydrophobic to hydrophilic molecules, thereby preventing them from diffusing freely across the lipid bilayer and necessitating a transport system like the sodium-dependent organic anion transporter (SOAT; SLC10A6). In addition, sulfated steroids can no longer bind to their nuclear receptors, rendering them biologically inactive. Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active forms. Data about the concentration of sulfated steroids in follicular fluid and the expression of the related enzymes are limited in horses and cattle. Recently, transcripts of the corresponding enzymes have been detected in cumulus cells of immature bovine cumulus-oocyte complex. Furthermore, it has been shown that small amounts of sulphated steroids are present in bovine follicular fluid. The objective of the study was to investigate the steroid metabolizing enzymes at the protein level via immunohistochemistry (IHC). Bovine ovaries collected at a slaughterhouse were categorized according to their oestrus cycle stage. Three pairs of ovaries of each cycle stage (proestrus, oestrus, postestrus, interestrus) were collected. Morphological criteria (ovaries: presence/absence of follicles and/or CL, size and number of follicles; closure of the cervix, amount of mucus) were employed to categorize them. Samples for IHC were fixed in formalin. After washing and dehydration, the samples were finally embedded in paraffin and mounted onto blocks. Indirect immunoperoxidase staining methods were applied using the streptavidin-biotin technique for signal enhancement following standard procedures. Tissue sections of 3 µm were mounted onto glass slides, and a polyclonal rabbit anti-human STS and a polyclonal rabbit anti-bovine SULT1E1 primary antibody were used to detect STS or SULT1E1, respectively. Negative controls were included using nonspecific rabbit IgG. The evaluation of the staining was descriptive in relation to a positive control (bovine placenta). Immunostaining for STS was detected in granulosa cells of antral and secondary follicles and in the endothelium of blood vessels, irrespective of the stage of the oestrus cycle. Moreover, staining for SULT1E1 was restricted to granulosa cells in antral follicles, again irrespective of the stage of the oestrus cycle. However, staining intensity for STS and SULT1E1 was only weak. These data indicate for the first time the presence of the steroid metabolising enzymes STS and SULT1E1 in bovine ovaries at the protein level. However, due to the low expression level, further studies are needed to clarify the function.
We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).
58 EFFECT OF LYSOPHOSPHATIDIC ACID ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IVF
Authors: Kim K.-J. , Lee E. , Hyun S.-H.
Published: December 2015

Lysophosphatidic acid (LPA) is a signalling molecule derived from phospholipid, known to have biological activities such as stimulating cell proliferation, differentiation, and migration. The purpose of this study was to investigate the effect of LPA on in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). We examined nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels and subsequent embryonic development after IVF in porcine oocytes matured in TCM-199 media containing either 0, 10, 30, or 60 μM LPA (No. of cultured oocytes 225, 225, 230, and 226, respectively). All data were compared for all groups using one-way ANOVA, followed by Duncan’s multiple range test. After 44 h of IVM, no significant difference was observed for nuclear maturation from the different (0, 10, 30, and 60 μM) LPA treatment groups (86.61, 87.48, 92.97, and 88.69%, respectively) compared with the control (P < 0.05). The 30 μM LPA treated group exhibited a significant (P < 0.05) increase in intracellular GSH levels and decrease in intracellular ROS levels compared with other LPA-treated groups. Oocytes matured with 30 μM LPA during IVM had significantly (P < 0.05) higher cleavage rates after PA (88.50%) than other LPA-treated groups (79.42, 83.87, and 83.65%, respectively). Blastocyst formation rates following PA increased up to 30 μM LPA, but the rates decreased in the 60 μM LPA-treated group (49.64, 53.28, 57.00, and 51.09%, respectively). However, a statistical difference was not observed (P < 0.05). Upon evaluation of embryonic competence following IVF, treatment with 30 μM LPA during IVM significantly increased cleavage rates (70.44%; P < 0.05) compared with 0 and 60 μM LPA-treated groups (61.51 and 65.27%, respectively). Blastocyst formation rates improved with increasing concentrations of LPA up to 30 µM; however, the rates decreased in the 60 μM LPA-treated group (26.34, 30.10, 33.26, and 27.77%, respectively). However, like the preceding PA blastocyst formation rates, a statistical difference was not observed (P < 0.05). In conclusion, treatment with 30 μM LPA during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, thereby decreasing the intracellular ROS level during oocyte maturation.
This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study from the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.
62 EFFECT OF PROGESTERONE ON CALCIUM REGULATION DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES
Authors: Choi Y.-K. , Kang H. Y. , Hwang J.-U. , Nam T. D. , Jeung E.-B.
Published: December 2015

During spontaneous cardiac differentiation of mouse embryonic stem cells (mESCs, cell line E14), the effect of progesterone on calcium regulation was investigated. Calcium (Ca2+) release from sarcoplasmic reticulum (SR) regulates various cellular functions including the smooth or skeletal muscle contraction. The cardiac L-type Ca2+ channel plays a key role in excitation-contraction coupling of cardiomyocytes and contraction-related gene expression. The mESCs formed mouse embryonic bodies (mEBs) by hanging-drop for 4 days, and mEBs were suspended for 2 days in differentiation medium; DMEM/F:12, 15% charcoal-dextran-treated FBS, β-mercaptoethanol, minimal essential medium NEAA, and penicillin/streptomycin. Then, mEBs were attached onto 6-well culture plates and differentiated into cardiomyocytes. We analysed mRNA expressions for the cardiac lineage markers and calcium-regulating genes. Percentage of beating mEBs was time-dependently increased during differentiation. Differentiated mEBs showed the highest beating ratio (92.11 ± 2.98%) after attachment for 12 days. Beating ratio was decreased to 64.86 ± 4.25% in progesterone-treated mEBs. The mRNA levels of cardiac markers such as Tbx20, Isl1, Foxh1, cTn1, and RyR2 were increased, and troponin protein was observed in beating mEBs via immunocytochemistry. Expression of calcium/contraction regulating genes including Trpv2, Ryr2, CaM2, and Mlck3 was down-regulated by progesterone treatment. These results indicate that progesterone has influences on cardiac differentiation and contraction of cardiomyocytes through regulating intercellular calcium ion.
This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.
64 EPITHELIAL MESENCHYMAL TRANSITION AND DIFFERENTIATION OF STEROIDOGENIC FACTOR 1 MOUSE EMBRYONIC STEM CELLS INTO THE STEROIDOGENIC CELLS
Authors: Kang H. Y. , Choi Y.-K. , Hwang J.-U. , Jeung E.-B.
Published: December 2015

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we transfected mouse embryonic stem cells (mESCs) with the mouse SF1 gene (SF1-mESCs) by using the nucleofector (Lonza), and selected SF1-mESCs by G418 250 μg mL–1. The selected cells were differentiated into granulosa-like cells through hanging-drops for 3 days, suspension culture for 1 day, then attachment onto 6-well plates. Expression of steroidogenesis-related genes and gonadal lineage-markers was analysed by real-time PCR. To test the phenotype for granulosa-like cells, transcripts of specific forkhead transcription factor (Foxl2) and follicle stimulating hormone receptor (Fshr) were measured. Also, expression of EMT-related genes, such as E-Cadherin (Cdh1), N-Cadherin (Cdh2), Snai1, Snai2 (Slug), Twist, and Vimentin, was monitored. SF1-mESCs were differentiated into the primitive streak‐mesendoderm and the steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (Hsd3b1), cytochrome P450-containing enzyme (Cyp)-11a1, and Cyp19a1 were time-dependently changed. Next, the mRNA levels of Foxl2 and Fshr representing granulosa-like cell were increased during differentiation of SF1-mESCs. Especially, the level of oestradiol and Cdh2 was increased at a specific differentiation time. We induced differentiation of mESCs into the functional granulosa-like cells through transfection of the mouse SF1 gene. These cells will be useful for further study and potential application of these cells in steroidogenesis.
This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.
65 EFFECT OF TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENT OF DONOR CELLS, FUSED EMBRYOS, OR BOTH ON THE DEVELOPMENTAL COMPETENCE, QUALITY, AND EPIGENETIC STATUS OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS
Authors: Saini M. , Selokar N. L. , Agrawal H. , Singla S. K. , Chauhan M. S. , Manik R. S. , Palta P.
Published: December 2015

Somatic cell nuclear transfer (SCNT) is a promising technology in buffalo for multiplication of elite animals, species conservation, and production of transgenic embryos for therapeutic applications. However, the cloning efficiency obtained in this species is very low, which might be due to improper reprogramming of donor cells after SCNT. Treatment of donor cells or fused embryos or both with epigenetic modifiers might be a suitable approach to improve the ability of donor cells to be reprogrammed. The present study was aimed at examining the effects of treatment of donor cells (24 h before SCNT) or fused embryos (10 h post-electrofusion) or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence, quality, and epigenetic status of buffalo embryos produced by hand-made cloning (HMC) as described earlier (Saini et al. 2014 Reprod. Fertil. Dev. doi: 10.1071/RD14176). The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher’s least significant difference test. The blastocyst rate was significantly higher (P < 0.05) and the apoptotic index was significantly lower (P < 0.05) in embryos produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls (Table 1). However, the cleavage rate and the total cell number were not significantly different among all the groups. The global level of H3K18ac, examined by immunofluorescence staining, was higher (P < 0.05) and that of H3K27me3 was lower (P < 0.01) in blastocysts produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls. These results show that treatment of donor cells, fused embryos, or both with TSA + 5-aza-dC improves the developmental competence and quality, and alters the epigenetic status of buffalo embryos produced by HMC. However, the effects of treatment with these epigenetic modifiers on the pregnancy rate require further studies.

Table 1.Effect of treatment of donor cells, fused embryos, or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence and level of apoptosis in cloned embryos
72 PRELIMINARY DATA ON THE PRESENCE OF BACTERIA IN THE UTERUS OF PREGNANT COWS
Authors: Pedersen H. G. , Knudsen L. R. V. , Agerholm J. S. , Jensen T. K. , Klitgaard K. S. , Karstrup C. C.
Published: December 2015

Bacterial invasion of the uterus during the postpartum period has been well described. Recent papers using 16S rRNA gene sequencing techniques suggest that the nonpregnant uterus contains a diverse flora of bacteria that are not necessarily pathogenic. In contrast, the pregnant uterus has until now been considered a sterile environment. The aim of the present study was to investigate whether bacteria were present in the uteri of pregnant cows. Uteri from pregnant, slaughtered animals (n = 47) were sampled. The surface of the uterus was wiped with alcohol, flame sterilized, and cut open with sterile scissors. Samples were taken from the endometrium and from the placentomes. The samples were embedded in paraffin, sectioned at 3 microns, and prepared for fluorescence in situ hybridization using a probe targeting the 16S rRNA of the domain bacteria, so that all bacteria regardless of species were visualised. Using fluorescence microscopy, the presence of bacteria within or on the surface of the endometrium and within the placentomes was noted. The stage of pregnancy was estimated to range from 26 to 263 days by measuring the size of the embryo or fetus. The endometrial samples from 85.1% (40/47) of pregnant cows contained bacteria. In 22 cows, the bacteria were localised within the endometrial tissue, whereas in the remaining 18 cows, the bacteria were on the epithelial surface. Placental samples were obtained from 43 cows, and 76.7% (33/43) of these contained bacteria. The presence of bacteria in the pregnant uterus may suggest that a cow can carry a pregnancy despite the presence of few potentially pathogenic bacteria or that normal flora exist in the uterus as in, for example, the vagina. In conclusion, bacteria were present in the endometrium and placentomes of pregnant cows. Further analyses using rRNA gene sequencing techniques will aim to confirm the presence of bacteria in the bovine pregnant uterus and to investigate which species of bacteria are present in the uterus during pregnancy.
85 CARBOXYETHYLGERMANIUM SESQUIOXIDE CONFERS ANTIOXIDATIVE PROTECTION FOR PORCINE IN VITRO-FERTILIZED EMBRYOS
Authors: Kim E. , Hyun S.-H.
Published: December 2015

Reactive oxygen species (ROS) are generated both from the environment as well as from within the embryos during normal embryo development. However, excessive production of ROS creates the defective effect on embryo development. The purpose of the present study was to determine the effect of antioxidant, carboxyethylgermanium sesquioxide (Ge-132), during in vitro culture (IVC) on embryonic development in porcine in vitro-fertilized (IVF) embryos. The different concentrations of Ge-132 (0, 100, 200, and 400 μg mL–1) were treated to the zygotes (n = 233, 234, 233, and 234, respectively). All of the experiments were repeated at least three times. To characterise putative porcine embryonic stem cell (ESC) derived from Ge-132 treated IVF blastocysts, several assays including an assessment of intensive alkaline phosphatase activity, embryoid body formation and expression analysis of pluripotency by immunofluorescence staining were performed. In the result of embryo developmental competence, the 100- and 200-μg mL–1 Ge-132 significantly (P < 0.05) increased cleavage rates (67.4% and 67.1%, respectively; P < 0.05) compared with the control group (61.6%). The 100 μg mL–1 Ge-132 had significantly (P < 0.05) higher blastocyst formation rates (32.6%) than the control group (26.5%). The Ge-132 supplement during IVC significantly (P < 0.05) increased GSH levels in embryos compared with those in the control, which led to the reduced ROS level. Two putative ESC lines were successfully derived from porcine IVF embryos cultured in 100 and 200 μg mL–1 Ge-132 treatment. The established putative porcine ESCs showed alkaline phosphatase activity, pluripotency-associated markers such as OCT4 and NANOG and the capacity for embryoid body formation, which confirm the differentiation potentials. Together, these results suggest that Ge-132 supplementations during IVC enhanced the developmental competence of porcine IVF embryos, most likely by increasing intracellular GSH synthesis, reducing ROS level, and it might help the derivation of putative porcine ESCs.
This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study by the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.
9 ENDOSCOPY-MEDIATED INTRATUBAL INSEMINATION IN THE COW – A PRELIMINARY REPORT ABOUT THE APPLICATION OF A NOVEL MINIMALLY INVASIVE INSEMINATION TECHNIQUE
Authors: Stein K. , Havlicek V. , Papp S. , Palm F. , Brem G. , Besenfelder U.
Published: December 2015

On their long path through the female reproductive tract to the fertilization site, spermatozoa are exposed to diverse influences and hazards of the cervical, uterine, and oviducal environment that naturally select viable sperms for the following fertilization. Consequently, this results in a reduction from several billions of sperms in the ejaculate to a functional sperm reservoir within the range of 102 in the isthmus of the Fallopian tube. A technique to deposit spermatozoa directly into the ampulla, thus bypassing most of the reproductive tract, enables a rigorous reduction in number of sperms deposited. Furthermore, it provides a direct assessment of sperm fertility. The aim of our study was to establish an endoscopy-assisted intratubal insemination technique using different sperm dosages, fresh or cryopreserved, to determine adequate conditions for optimal fertilization. Eighteen Simmental heifers were inseminated with fresh semen, and 9 heifers were inseminated with frozen semen using this novel technique. The heifers were synchronized using a modified Ovsynch protocol, and insemination was conducted 18 to 20 h after the second gonadotropin-releasing hormone application. Insemination of heifers was performed under epidural anaesthesia. A tubing system bearing the endoscope and an insemination device was introduced through the vaginal wall into the peritoneal cavity. The insemination device consisted of a tube connected to a curved glass capillary tube loaded with semen. After a visual examination of the ovaries for the presence of an ovulatory Graafian follicle, the capillary tube was inserted directly via the infundibulum into the ipsilateral ampulla and the semen dose was deposited. The entire procedure took ~10 min. Two days later the oviduct was flushed by the same technique. A tubing system connected to a metal catheter served for flushing the embryos and unfertilized oocytes from the oviduct into the uterine horn. Afterward, embryos and oocytes were collected by flushing the uterine horn using an embryo flushing catheter and an embryo filter (EmCon). Embryos were stained using a Hoechst dye to visualise the numbers of attached spermatozoa to the zonae pellucidae. From 18 inseminations with fresh semen doses of 7 to 28 million sperms, 7 embryos at the 2- to 8-cell stage were found. Two of these embryos had more than 10 accessory sperms (AS), 3 had 3 to 6 AS, and 2 were without AS. From 9 inseminations with frozen semen doses containing 1.5 million sperms, we obtained 2 embryos, one at the 4-cell stage without AS and one at the 8-cell stage with 5 AS. Additionally, 3 unfertilized oocytes were collected. In conclusion, these preliminary results demonstrate a promising technique for intratubal AI, which has to be further optimized by studying numbers and treatment of spermatozoa and time of insemination.
90 LIPID CONTENT OF IN VIVO- AND IN VITRO-PRODUCED JERSEY AND HOLSTEIN CATTLE EMBRYOS AND THE EFFECT OF FORSKOLIN ON EMBRYO LIPID REDUCTION
Authors: Rhodes-Long K. , Campos-Chillon L. F. , Barceló-Fimbres M. , Altermatt J. L.
Published: December 2015

Jersey embryos have been suggested to have higher lipid content and lower tolerance to cryopreservation. In addition, in vitro-produced (IVP) bovine embryos have darker cytoplasm as a consequence of higher lipid accumulation than in vivo-derived embryos, associated with impaired embryo quality and reduced cryotolerance. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. We hypothesised that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin would reduce lipid content of IVP embryos. The objectives of this experiment were (1) to analyse lipid content of in vivo and IVP Jersey and Holstein cattle embryos and (2) to evaluate the effect of forskolin added to IVP culture media. The factorial experimental design used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forskolin). IVP embryos (n = 27 blastocysts) were collected from super-stimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of forskolin (10 µM) at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data (Table 1) were analysed by ANOVA, and means were compared using Tukey’s HSD. Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. Addition of forskolin to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. Further studies including cryopreservation and transfer of IVP + forskolin embryos to recipients are necessary to corroborate the findings of the present study.

Table 1.Fluorescence intensity of in vivo-produced and IVP Jersey and Holstein embryos
91 EMBRYO SUGAR CONSUMPTION DURING IN VITRO DEVELOPMENT
Authors: Rubessa M. , Ambrosi A. , Polkoff K. M. , Denmark S. E. , Wheeler M. B.
Published: December 2015

Despite the presence of glycogen and triglycerides as energy reserves in bovine embryos, the viability of embryonic cells in culture is maintained primarily by energetic substrates present in the culture medium. The aim of this experiment was evaluate the embryo metabolism during three important stages of development: Days 1 to 3, Days 3 to 5, and Days 5 to 7; before the formation of Barr body, before the differentiation into ICM and trophectoderm, and during the blastocyst formation, respectively. In this study, we evaluated some energetic substrates (Krebs cycle intermediates). For these analyses we chose to use proton magnetic resonance (H1-NMR) spectroscopy. Matured oocytes were purchased from DeSoto Biosciences (Seymour, TN, USA). The matured cumulus-oocyte complexes were fertilized in vitro according to our standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). For each group (Days 1–3, 3–5, and 5–7), the zygotes were place in individual drops, and after 48 h of culture, they were placed into well of the well (WOW) culture. For the 3–5 and 5–7 groups, the zygotes were first place in 400 μL of SOF and then moved in a single drop. The 5–7 group was not placed in the WOW system. Zygotes were incubated in a humidified mixture of 5% CO2, 6% O2, and 88% N2 in air at 39°C. The embryos were scored for quality on the basis of morphological criteria. Samples of media (40 μL) were added to 560 μL of a stock solution prepared by dissolving 5.0 mg of sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate (TSP) in 50 mL of deuterium oxide. The TSP acted both as a chemical shift reference and as an internal standard for the purposes of quantitation. The resulting diluted samples were transferred to a 5-mm NMR tube. Samples were analysed on a Varian VNS-750 NB (750 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA). A total of 30 embryos were evaluated, 10 per stage. Data were statistically analysed through an analysis of variance using the Generalized Linear Model (GLM) procedure (SPSS, version 18, 2009). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α level was set at 0.05. All data were expressed as quadratic mean and mean standard error. The results, reported in Table 1, show that there was no statistical difference when we compare the lactate, myo-inositol, and pyruvate concentrations. However, there were significant differences when we focused on acetate and formate levels: after Day 3 the production of these two substrates significantly increased. Formate and acetate are important metabolites of the Krebs cycle. The changes of these two substrates are important signs that embryos start glycolysis after Day 3.

Table 1.Quadratic means and mean standard errors of formate, lactate, myo-inositol, pyruvate, and acetate for Day 1–3, 3–5, 5–7, and control groups
99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS
Authors: Lopera-Vasquez R. , Hamdi M. , Maillo V. , Nunez C. , Yanez-Mo M. , Ramirez M. A. , Gutierrez-Adan A. , Bermejo-Alvarez P. , Rizos D.
Published: December 2015

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo.
Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).
Catch Rates and Cost Effectiveness of Entrapment Gears for Asian Carp: A Comparison of Pound Nets, Hoop Nets, and Fyke Nets in Backwater Lakes of the Illinois River
Authors: Collins Scott F. , Butler Steven E. , Diana Matthew J. , Wahl David H.
Published: December 2015

We compared three entrapment gears to determine which method was the most effective for capturing invasive Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix in terms of numbers of fish captured and labor invested. Gears were deployed concurrently in two backwater lakes of the Illinois River during the summers of 2012–2014. Overall, the nightly catch rates of all fishes, Bighead Carp, and Silver Carp were one to three orders of magnitude greater in pound nets than in either fyke nets or hoop nets. Pound nets collected larger Bighead Carp than hoop nets and fyke nets. Hoop nets were ineffective at catching Asian carp in backwater lakes. Estimation of the effort required to deploy, maintain, and remove each gear type indicated that pound nets were the most cost-effective gear due to their high catch rates of Asian carp relative to the labor hours invested to collect the catch. Pound nets appear to be an effective means of removing Asian carp in backwater lake habitats of the Illinois River.

Received April 20, 2015; accepted September 3, 2015
Rings of invariants for modular representations of the Klein four group
Authors: Sezer Müfit , Shank R.
Published: December 2015

We study the rings of invariants for the indecomposable modular representations of the Klein four group. For each such representation we compute the Noether number and give minimal generating sets for the Hilbert ideal and the field of fractions. We observe that, with the exception of the regular representation, the Hilbert ideal for each of these representations is a complete intersection.
Epithelial Sel1L is required for the maintenance of intestinal homeostasis
Authors: Sun Shengyi , Lourie Rohan , Cohen Sara B. , Ji Yewei , Goodrich Julia K. , Poole Angela C. , Ley Ruth E. , Denkers Eric Y. , McGuckin Michael A. , Long Qiaoming , Duhamel Gerald E. , Simpson Kenneth W. , Qi Ling
Published: December 2015

Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)–associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn’s disease (CD). Consistent with this observation, laboratory mice with enterocyte-specific Sel1L deficiency (Sel1LΔIEC) develop spontaneous enteritis and have increased susceptibility to Toxoplasma gondii–induced ileitis. This is associated with profound defects in Paneth cells and a disproportionate increase of Ruminococcus gnavus, a mucolytic bacterium with known association with CD. Surprisingly, whereas both ER stress sensor IRE1α and effector CHOP are activated in the small intestine of Sel1LΔIEC mice, they are not solely responsible for ERAD deficiency–associated lesions seen in the small intestine. Thus our study points to a constitutive role of Sel1L-Hrd1 ERAD in epithelial cell biology and the pathogenesis of intestinal inflammation in CD.
Sensitization and Serological Biomarkers in Knee Osteoarthritis Patients With Different Degrees of Synovitis
Authors: Petersen Kristian K. , Siebuhr Anne S. , Graven-Nielsen Thomas , Simonsen Ole , Boesen Mikael , Gudbergsen Henrik , Karsdal Morten , Bay-Jensen Anne C. , Arendt-Nielsen Lars
Published: December 2015

Objective:

Synovitis is a frequent condition in knee osteoarthritis (KOA) and has been associated with pain. This study investigated the links between the pressure hyperalgesia, the clinical pain, the degree of the synovitis, inflammatory biomarkers, and tissue-specific biomarkers in KOA patients.

Materials and Methods:

Fifty-eight KOA patients and 33 pain-free controls participated in this study. The patients were magnetic resonance imaging scanned, and the Boston-Leeds OA Knee Score (BLOKS, 0 to 3) was used to assess the degree of synovitis. The maximal knee pain intensity over the last 24 hours was scored on a visual analog scale (VAS). The pressure pain thresholds (PPTs) were assessed over the KOA-affected knee. Serological biomarkers were measured in fasting serum: high-sensitive C-reactive protein, matrix metalloproteinase–mediated degradation of CRP, and matrix metalloproteinase–mediated collagen type I, II, and III degradation (C1M [connective tissue], C2M [cartilage], C3M [synovial membrane]).

Results:

Compared with controls, the KOA patients showed increased levels of C1M (P<0.02), C2M (P<0.001), and high-sensitive C-reactive protein (P<0.02), decreased level of C3M (P<0.03), and reduced PPTs (P<0.03). Patients with no (BLOKS 0) and moderate to severe (BLOKS 2&3) synovitis had significantly lower PPTs compared with mild synovitis (BLOKS 1). Significantly negative correlations were found between VAS and PPTs. No correlations were found between BLOKS and the VAS, PPT, or biomarkers.

Discussion:

Patients without and with moderate to severe synovitis demonstrated local pressure hyperalgesia and increased degrees of: (1) systemic inflammation, (2) connective tissue degradation, (3) cartilage degradation, and (4) decreased synovial membrane degradation as compared with controls.
The Fate of the Neuromuscular Hip After Spinal Fusion
Authors: Crawford Lindsay , Herrera-Soto Jose , Ruder John A. , Phillips Jonathan , Knapp Raymond
Published: December 2015

Background:

The goals of treatment of neuromuscular scoliosis are to achieve a balanced spine and level pelvis with most constructs including pelvic fixation. However, the pelvis can become a stiff “end vertebra” that prevents compensatory mechanisms to adjust to hip deformities in this patient population. The purpose of this study is to determine the frequency of hip pathology and surgery after spinal fusion in this patient population.

Methods:

We performed a retrospective chart and radiographic review of cerebral palsy patients who underwent posterior spinal fusion (PSF) at our institution from 2005 to 2011. We collected radiographic data of preoperative and postoperative pelvic obliquity and hip reduction status and position (up, level, down). We further evaluated patients requiring hip surgery (containment or salvage).

Results:

Of 47 patients with an average follow-up of 3.5 years after spinal fusion, 21 (45%) underwent a hip procedure. Thirty-eight patients (81%) demonstrated or developed hip subluxation/dislocation. Hip pathology occurred more often in the up hip, but the pathologic down hip more often underwent a hip surgery. Eight new hip subluxation/dislocations occurred after spine surgery. Three (38%) of the new postoperative subluxation/dislocations required hip surgery; all had pelvic obliquity <6 degrees. Eleven patients underwent hip surgery before PSF, 7 were varus femoral osteotomies for subluxation, whereas 5 hips required salvage. In follow-up after PSF, none of these had a new dislocation. Ten patients required hip surgery after PSF at a mean of 1.6 years after PSF. Eight patients had a salvage procedure for painful hip and 2 varus femoral osteotomies for subluxations.

Conclusions:

In our cerebral palsy patients who underwent PSF, 45% of these patients required a hip procedure. In the patients who had containment before PSF, the hips maintained reduction after spinal fixation. After correction of pelvic obliquity, 17% of patients had new-onset hip subluxation/dislocation after PSF. Postoperative subluxation/dislocation was not dependent on whether the hip was up or down preoperatively.

Level of Evidence:

IV, Retrospective.
Influence of primary care professionals on early detection of breast cancer: different perception between family physicians and nursing professionals
Authors: Esteban-Vasallo María D. , Aerny-Perreten Nicole , García-Riolobos Carmen , López Rubio Aranzazu , Domínguez-Berjón Felicitas
Published: December 2015

The aim of this study was to assess the level of self-perceived influence on early detection of breast cancer among nurses and family physicians (FP) working in primary care and to identify the factors associated with a lower perception in each group. In the Autonomous Community of Madrid, an online questionnaire on cancer prevention was sent to a random sample of primary care professionals (3586 FP and nurses). The data included sex, age, profession, years in primary care, specific postgraduate training, and opinions and attitudes toward cancer prevention. A descriptive analysis was carried out. Factors associated with a low/null self-perceived influence on early detection of breast cancer were analyzed separately for FP and nurses with multivariate logistic regression models. In all, 76.4% of the participants considered that their influence on the early detection of breast cancer was enough/high. FP attributed to themselves a higher influence than nurses (enough/high: 86.0 vs. 64.6%, P<0.01), and a lower perception was associated with a low/null perceived utility of their individual intervention [odds ratio (OR): 6.42, 95% confidence interval (CI) 2.77–14.85]. Among nurses, also associated with this low/null perceived utility [adjusted odds ratio (aOR): 2.81, 95% CI 1.37–5.77] were the absence of postgraduate training in the matter (aOR: 2.56, 95% CI 1.05–6.25), and a low/null perception of feasibility of prevention programs in their centers (aOR: 1.93, 95% CI 1.25–2.98). Primary care professionals perceive their activity in the early detection of breast cancer as relevant, especially FP. To increase knowledge of the utility of individual interventions, specific training and improvement of the feasibility of these programs could enhance this perception.