bluuebunny/crossref_metadata_2025 huggingface dataset:
Pole walking down‐under: profile of pole walking leaders, walkers and programs in Australia and factors relating to participation
Authors: Fritschi Juliette O. , van Uffelen Jannique G. Z. , Brown Wendy J.
Published: December 2014

Issue addressed

Although pole walking (PW) has the potential to be a useful health‐enhancing physical activity (PA), little is known about by whom or how it is being practised. The aims of this study were to describe (1) the characteristics of PW leaders, pole walkers and PW programs in Australia, and (2) participants' perceptions of PW and their reasons for participation.

Methods

In 2012, PW leaders (n = 31) and walkers (n = 107) completed self‐administered surveys that included questions about participants' sociodemographic and health characteristics, PW programs and perceptions of PW. Data were analysed using SPSS.

Results

Leaders and walkers were generally born in Australia (leaders, 71%; walkers, 83%), older (leaders, 55 years [s.d. 11.5]; walkers, 65 years [s.d. 10.6]) and female (leaders, 77%; walkers, 79%). Most walkers (82%) walked regularly in groups, approximately once per week for about an hour, at light to moderate intensity. The program's aims most strongly endorsed by PW leaders were to increase participant enjoyment (90%), increase PA levels (81%), provide a positive social experience (77%) and increase PA confidence (71%). The most strongly endorsed motivations for PW among walkers were to remain physically active (63%), improve fitness (62%) and personal and social enjoyment (60%).

Conclusions

In Australia, PW is being practised by a health conscious, older population. It is perceived as an enjoyable and healthenhancing outdoor activity.

So what?

Health and exercise practitioners may find that PW is a beneficial form of PA for older Australians.
Genes and environmental factors that influence disease resistance to microbes in the female reproductive tract of dairy cattle
Authors: Sheldon I. M.
Published: December 2014

Microbes commonly infect the female reproductive tract of cattle, causing infertility, abortion and post partum uterine diseases. When organisms reach the uterus, the resistance to disease depends on the balance between the classic triad of the virulence of the microbes, the host defence systems and the environment. The present review considers each aspect of this triad, using postpartum uterine disease as an exemplar for understanding disease resistance. The bacteria that cause postpartum uterine disease are adapted to the endometrium, and their microbial toxins cause tissue damage and inflammation. However, non-specific defence systems counter ascending infections of the female reproductive tract, and inflammatory responses in the endometrium are driven by innate immunity. Disease resistance to bacterial infection involves many genes involved in the maintenance or restoration of tissue homeostasis in the endometrium, including antimicrobial peptides, complement, cytokines, chemokines and Toll-like receptors. The most important environmental factors facilitating the development of postpartum uterine disease are related to trauma of the reproductive tract and to the metabolic stress of lactation in dairy cows. Long-term solutions for uterine disease will include genetic selection for disease resistance and optimising the care of the animal before, during and after parturition.
Metabolic determinants of embryonic development and stem cell fate
Authors: Folmes Clifford D. L. , Terzic Andre
Published: December 2014

Decoding stem cell metabolism has implicated a tight linkage between energy metabolism and cell fate regulation, a dynamic interplay vital in the execution of developmental and differentiation programs. The inherent plasticity in energy metabolism enables prioritisation of metabolic pathways in support of stage-specific demands. Beyond traditional support of energetic needs, intermediate metabolism may also dictate cell fate choices through regulation of cellular signalling and epigenetic regulation of gene expression. The notion of a ‘metabolism-centric’ control of stem cell differentiation has been informed by developmental embryogenesis based upon an on-demand paradigm paramount in defining diverse developmental behaviours, from a post-fertilisation nascent zygote to complex organogenesis leading to adequate tissue formation and maturation. Monitored through natural or bioengineered stem cell surrogates, nutrient-responsive metabolites are identified as mediators of cross-talk between metabolic flux, cell signalling and epigenetic regulation charting, collectively, whether a cell will self-renew to maintain progenitor pools, lineage specify to ensure tissue (re)generation or remain quiescent to curb stress damage. Thus, bioenergetics are increasingly recognised as integral in governing stemness and associated organogenic decisions, paving the way for metabolism-defined targets in control of embryology, stem cell biology and tissue regeneration.
102 SUPPLEMENTATION WITH ESTRADIOL CYPIONATE AT THE ONSET OF A SYNCHRONIZED PROESTRUS ALTERS THE UTERINE GENE EXPRESSION OF SUCKLED ANESTROUS BEEF COWS
Authors: Sá Filho M. F. , Pugliesi G. , Gonella-Diaza A. M. , Sponchiado M. , Mendanha M. F. , Ramos R. S. , Andrade S. C. S. , Gasperin G. , Goissis M. D. , Mesquita F. S. , Coutinho L. L. , Baruselli P. S. , Binelli M.
Published: December 2014

The present study evaluated the effect of oestradiol cypionate (ECP) supplementation at the onset of a proestrus [i.e. at progesterone (P4) device removal of the synchronization of ovulation protocol] on the global endometrial gene expression of suckled anestrous beef cows. A total of 12 suckled cows presenting absence of corpus luteum detected by ultrasound received 2 mg of oestradiol benzoate IM and an intravaginal P4 device. Eight days later, P4 devices were removed, and the cows received 500 mg of sodium cloprostenol IM. Cows were blocked according to body condition score and diameter of largest follicle (LF) at P4 device removal and were randomly assigned to receive an IM treatment with 1 mg of ECP (ECP, n = 6) or not (CON, n = 6) at the P4 device removal. All cows received 10 μg of buserelin acetate 48 h after P4 device removal and were timed AI immediately after. Cows presenting a new corpus luteum formed 6 days after the gonadotropin-releasing hormone treatment had an endometrial fragment collected by transcervical biopsy. RNA-Seq analysis was performed and the transcriptome profile was obtained. The significance of differential gene expression was assessed with the package DESEqn 2 v.1.2.10 and the differential expression estimation was based on the GLM followed by the Wald test. The significance threshold was established as an FDR- Benjamini-Hochberg-adjusted P-value of <0.1. The integrated analysis was performed using DAVID database. In total, 135 transcripts were differentially expressed between ECP and CON groups, of which 73 genes were up-regulated by ECP supplementation and 62 genes were up-regulated in the CON cows. Two pathways were overrepresented by the ECP-induced transcripts: pathways in cancer (n = 5 genes) and small cell lung cancer (n = 3 genes). On the other hand, ECP-inhibited transcripts indicated the enrichment of 3 pathways: Parkinson's disease (n = 3 genes), oxidative phosphorylation (n = 3 genes), and Alzheimer's disease (n = 3 genes). More specifically, ECP-induced transcripts associated with pathways in cancer [gene symbol (fold change); respectively] were LAMC3 (1.55), PTCH1 (1.51), PTCH2 (1.52), PIK3R3 (1.22), and PIAS1 (1.18), whereas ECP-inhibited transcripts associated with oxidative phosphorylation were ATP5F1 (1.18), ATP5J (1.24), and NDUFB3 (1.37). Therefore, ECP supplementation at onset of the synchronized proestrous slightly alters the uterine transcriptome. The enriched pathways affected by the ECP supplementation described in this work need to be studied more but these results show candidate pathways that can be associated with uterine environment and receptivity and with possible regulation by oestradiol supplementation given at the onset of the proestrous.
109 EXPRESSION OF GENES RELATED TO ENDOMETRIAL RECEPTIVITY IN EQUINE ENDOMETRIUM DURING THE ESTROUS CYCLE AND EARLY PREGNANCY
Authors: Hitit M. , Guzeloglu A. , Ozel C. , Atli M. O. , Kurar E. , Kayis S. A.
Published: December 2014

A set of genes that display differential expression levels in the reproductive tract could serve as beneficial markers of endometrial receptivity. SERPINA14 is present in the uterus during pregnancy and suppresses lymphocyte accumulation. Osteopontin is the ligand of integrin β3 and enables trophoblast communication during implantation. Leukemia inhibitory factor (LIF) is involved in inflammatory cell signalling and contributes to implantation by regulating immune cells. The objective was to assess the expression of SERPINA14, osteopontin, and LIF mRNAs in the equine endometrium during the oestrous cycle and early pregnancy. Biopsies were obtained from mares on day of ovulation (d 0, n = 4), late diestrus (LD, n = 4, high progesterone [P4]), and after luteolysis at the beginning of oestrus phase (AL, n = 4, <1 ng mL–1 P4) of the cycle. Biopsies were also taken on days 14 (P14, n = 4), 18 (P18, n = 4), and 22 (P22; n = 4) of pregnancy. Relative mRNA expression levels of genes were quantified using real-time quantitative RT-qPCR in duplicate. Data were analysed using one-way ANOVA, and l.s.d. test was applied. Both the oestrous cycle and early pregnancy increased SERPINA14 mRNA levels compared to d0. Expression of LIF mRNA was not significantly regulated except for a decline at AL. Expression of osteopontin mRNA was up-regulated during the oestrous cycle at LD while early pregnancy inhibited this up-regulation. The results suggest that the genes studied related to endometrial receptivity are strictly regulated accordingly to the stage of oestrous cycle, probably by circulating ovarian steroids, specifically progesterone, and pregnancy-associated factors are also involved in this regulation.
This project was partially funded by TUBITAK 107O035 to AG and DUBAP 14VF12 to MOA. MH was supported by OYP 2013-090.
128 ADDING SERUM OF COWS SUPPLEMENTED WITH β-CAROTENE DURING BOVINE IN VITRO EMBRYO CULTURE HAS NO EFFECT ON EMBRYO DEVELOPMENT
Authors: De Bie J. , Merckx E. , Andries S. , Immig I. , Bols P. E. J. , Leroy J. L. M. R.
Published: December 2014

Elevated serum NEFA concentrations, typically present in negative energy balance (NEB) cows, are known to compromise bovine in vitro oocyte and embryo quality and developmental competence. These observations seem to be associated with oxidative stress. Therefore, antioxidant supplementation such as β-carotene (bC) can be a promising solution to ameliorate embryo quality and survival. However, little is known about the possible neutralizing effect of bC on NEB-compromised embryos. Accordingly, we hypothesise that bC can overcome the potential negative effects of metabolic conditions associated with NEB on embryo development. To investigate this we aimed to evaluate the effect of serum from bC-supplemented positive energy balance (PEB) or NEB cows on embryonic developmental competence. A total of 5 nonlactating Holstein-Friesian cows were subjected to 4 consecutive dietary treatments, 28 days each: 1) 1.2 × maintenance (M) (= PEB–bC), 2) 1.2 × M with daily 2000 mg of bC (Rovimix 10% bC, DSM) (= PEB+bC), 3) 0.6 × M + bC (= NEB+bC), and 4) after a 6 week acclimatization period 0.6 × M (= NEB–bC). Serum was collected 72 h after ovulation, pooled per dietary treatment, and heat inactivated during 30 min at 56°C. In total 1404 bovine slaughterhouse grade 1 cumulus-oocyte complexes were serum-free matured (4 repeats), routinely fertilized, and cultivated for 6.7 days with the addition of 10% serum of the 4 different treatments. Cleavage (48 h post-insemination), blastocyst rates (7.7 days post-insemination), and the rates of blastocysts from cleaved zygotes were calculated. Developmental competence data were compared between the 4 treatments using a binary logistic regression model taking replicate, treatment, and the interaction of both factors into account. The NEFA and bC data were analysed using a paired-samples t-test (IBM SPSS Statistics 20). Bonferroni correction was applied. Serum NEFA concentrations were significantly elevated in NEB compared to PEB (0.36 ± 0.18 mM v. 0.21 ± 0.11 mM; P = 0.011). β-Carotene supplementation drastically increased bC concentrations in serum in NEB (0.44 ± 0.18 μg mL–1 v. 3.28 ± 0.78 μg mL–1; P < 0.001) as well as in PEB (1.02 ± 0.91 μg mL–1 v. 3.04 ± 1.28 μg mL–1; P < 0.001). Unexpectedly no significant differences were found on cleavage rates (on average 81%), subsequent development until blastocyst stage (on average 29%), or blastocyst rates from cleaved zygotes (on average 36%). Briefly, our model was not able to indicate any negative effect of NEB serum on in vitro embryo development compared with PEB, and hence no extra beneficial effects of bC could be observed on the outcome. In conclusion, these data show that more research is needed to optimize this model to investigate the effect of specific dietary strategies on pre-implantation embryo quality.
144 PREGNANCY RATES FOLLOWING THE TRANSFER OF IN VITRO FERTILIZED EMBRYOS TO RECIPIENTS ON DAY 6, 7, OR 8 AFTER OESTRUS
Authors: Fry R. C. , Fry K. L. , McCartney H. A. , Geddes W. R. , Geddes K.
Published: December 2014

The aim of this experiment was to investigate the effect of day of synchrony on the pregnancy rate of recipients following the transfer of Day 7 IVF embryos. In addition, the effect of IVF embryo grade and corpus luteum (CL) grade of recipients was determined. A total of 317 cumulus-oocyte complexes collected from 24 dry Brahman cows by TVR were matured, fertilized, and cultured under standard in vitro production procedures (Fry et al. 2003 Theriogenology 59, 446). A total of 89 (44 Grade 1, 43 Grade 2, and 2 Grade 3, IETS classification) in vitro-produced embryos were transferred to parous 4- to 9-year-old dry Brahman cross recipient cattle 7 days after IVF. Two groups of recipient cows were synchronised one day apart with an 8-day CIDR/pg protocol so that oestrous would be concentrated over 3 days with the middle day aligning with the day of IVF (Day 0). Donors that produced a large number of IVF embryos had these divided and transferred into recipients either on Day –1 or Day +1 of synchrony, and those producing less than 4 IVF embryos were transferred into recipients on Day 0. At embryo transfer the ovaries of the recipient were palpated and then scanned by rectal ultrasound and the grade of CL noted (Grade 1 = large distinct CL by palpation, Grade 2 = small distinct CL by palpation, Grade 3 = CL not distinguishable by palpation). Pregnancy was diagnosed by ultrasound scanning on Day 92. Although recipient numbers were low, differences in pregnancy rate between groups were analysed by Chi-squared. Data from the 2 Grade 3 embryos transferred were not included in the analysis (0/2 pregnant). Similar (P > 0.05) pregnancy rates were found when Day 7 IVF embryos were transferred to either Day 6 (17/32 = 53%), Day 7 (9/24 = 38%), or Day 8 (14/31 = 45%) recipients. Furthermore, neither the grade of the embryo (Grade 1: 20/44 = 45%, Grade 2: 20/43 = 47%) nor the grade of recipient CL (Grade 1: 17/45 = 38%, Grade 2: 17/29 = 59%, Grade 3: 6/13 = 46%) effected pregnancy rate (P > 0.05). This experiment demonstrates the flexibility of the IVF embryo to achieve an acceptable pregnancy rate over a range of recipient stages thereby allowing a high usage rate of good-quality recipients in an IVF embryo transfer program.
147 SOFTWARE FOR MORPHOLOGICAL CLASSIFICATION OF CATTLE BLASTOCYSTS
Authors: Matos F. D. , Rocha J. C. , Nogueira M. F. G.
Published: December 2014

Embryonic morphological classification has great importance for numerous laboratory techniques (from basic to applied research in assisted reproduction). However, the method used to perform the classification of embryos in varying degrees of quality has always been based on the subjectivity of the evaluator. Although quality standards and descriptions of morphological characteristics that categorize an embryo in each grade are established, currently there is not an accurate method that can generate consistent and reliable results. Thus, our work resulted in the development of software able to perform the classification of morphological quality of bovine blastocysts. Artificial intelligence techniques (such as artificial neural networks) were used in the development. Results indicate an overall accuracy of 79.2% in the classification of bovine blastocysts in 3 degrees of quality. For blastocysts classified as excellent or good (class 1), the hit rate is 82.6%; for blastocysts classified as regular (class 2), the hit rate is 16.7%; and for blastocysts classified as poor (class 3), the hit rate is 91.7%.
157 EFFECTS OF METABOLIC CONDITIONS OF DAIRY COWS DURING TRANSITION PERIOD AND EARLY LACTATION IN WINTER AND SUMMER ON HEALTH AND FERTILITY
Authors: Maturana Filho M. , Lemes K. M. , Naves J. R. , Santin T. , Nishimura T. K. , Madureira E. H.
Published: December 2014

The aim of this study was to investigate and determine which metabolic predictors measured during the transition period, have a better association with health and fertility parameters in dairy cows in winter (W) and summer (S).The experiment was conducted with 235 multiparous Holstein dairy cows. The animals were divided, retrospectively into the experimental groups [High Production (AP), ≥45.9 to 65 kg of milk during peak lactation; Medium production (MP), between 30 and 45.8 kg of milk during peak lactation]. Blood samples and evaluations occurred during the transition period and during the timed fixed artificial insemination (TAI). Data were analysed for the main effects of group, day, and their interaction using the PROC MIXED procedure of SAS software (SAS 9.3, SAS Institute, Inc., Cary, NC, USA). Creatine kinase (CK) plasmatic concentrations increased during peak lactation in all groups, but the difference was not significant. Variations in superoxide dismutase (SOD) concentrations were observed in HP cows in both seasons (HPW and HPS). These cows had higher levels of superoxide dismutase (SOD) when compared of MP cows (MPW and MPS), with significant changes since 7 days before calving and remained higher until 104 days of lactation. The differences were observed in lipid profile, particularly in NEFA concentration. For this variable, there were group (P < 0.001) and day (P < 0.001) effects and also double interactions (day v. season, P < 0.001) and triple interactions (group v. day v. season, P = 0.009). The HPS cows had higher values, as well as a variation pattern of urea concentration (P < 0.05), different from the other groups until 100 days in milk (DEL). No differences were observed in diseases incidences between group or periods (P > 0.05). There was a statistical tendency to all groups in the summer season for earlier calving (P = 0.1), less calf weight (P = 0.07), and retained placenta (P = 0.09) and metritis incidence (P = 0.08). These parameters were strongly correlated with metritis disease incidence and the significant parameters: Earlier calving (P = 0.03), dystocia (P = 0.05), and retained placenta (P = 0.009). The NEFA (0.4 mmol L–1) and BHBA concentrations (0.7 mmol L–1) were highly correlated with the occurrence of uterine diseases. We observed high levels of AST (around 128 U/L) pre calving in ketotic and in cows with displacement of abomasum. According to these results, we concluded that milk production was not a risk factor for fertility in the first three services. Also, metabolic changes in the transition period were determinant in milk yield and health diseases.
Research was supported by FAPESP, CNPq, CAPES, Fazenda Colorado, and Ourofino animal health.
163 RE-ESTABLISHING REPRODUCTIVE CYCLICITY IN A FEMALE ASIAN SMALL-CLAWED OTTER FOLLOWING SUPRELORIN-INDUCED CONTRACEPTION: A CASE STUDY
Authors: Bateman H. L. , Swanson W. F.
Published: December 2014

Asian small-clawed otters (ASCO) are a popular species to exhibit in zoological institutions globally, and are found in managed populations in the United States, Europe, Asia, and Australia. Captive breeding of these otters is integral to population sustainability, with management programs using pedigree analyses to make specific breeding recommendations to ensure long-term genetic viability. Because of the familial social structure of ASCO and limited space within zoos, physical separation of animals is not always possible for temporary breeding prevention, and short-term contraception may be preferred. In US zoos, Suprelorin (deslorelin; Virbac Australia, Milperra, Australia), a GnRH agonist, has been recommended for contraception of female carnivores, due to its small implant size and lack of side effects often associated with hormone-based contraceptives. However, the duration of reproductive suppression with Suprelorin may be excessive and reversibility (i.e. resumption of cyclicity, ovulation, and pregnancies) in implanted females across a range of species has been variable, unpredictable, and prolonged. Since 2006, 49 female ASCO have been implanted with Suprelorin at least once, and, of these, only two females have shown confirmed reversibility with pregnancies. No ASCO females implanted more than once have thus far exhibited reversibility (personal communication, AZA Wildlife Contraceptive Center, St. Louis, MO, USA). In this case study, fecal hormone monitoring of one female ASCO implanted with Suprelorin three times (Dec 2007, Jan 2009, Mar 2010), showed a lack of ovarian cyclicity and ovulation during the three years since her last implant. In an attempt to induce ovarian follicular growth and ovulation, this female was injected (IM) with exogenous gonadotropins (100 IU of equine chorionic gonadotropin followed 80 h later with 3000 IU of porcine luteinizing hormone). Fecal progesterone monitoring confirmed ovulation followed by a 76 day pseudopregnancy, with temporal characteristics similar to those previously reported in naturally cycling ASCO (Bateman et al. 2009 Zoo Biol. 28, 107–126). Following the induced pseudopregnancy and ~55 days of basal progestin levels, this female was observed breeding with a cohabitating ASCO male. Fecal hormone monitoring revealed subsequent ovulation and the occurrence of another pseudopregnancy of normal duration. These preliminary findings suggest that exogenous gonadotropin treatment may be useful for promoting resumption of normal ovarian cyclicity and ovulatory responses in ASCO following prolonged reproductive suppression with Suprelorin.
Corrigendum to: 18 COMPARISON BETWEEN THE EFFICIENCY OF 30-mg FLUROGESTONE ACETATE INTRAVAGINAL SPONGE (FGA-30) AND CONTROLLED INTERNAL DRUG RELEASE (CIDR) TO SYNCHRONIZE OESTRUS IN EWES
Authors: Swelum A. , Al-Owaimer A. , Abouheif M.
Published: December 2014

The aim of the present study was comparing between the efficiency of FGA-30 (intravaginal polyurethane sponges impregnated with 30 mg of flurogestone acetate; Synchropart®, Ceva Sante, Animale, France) and EAZI-Breed™ CIDR® (an inert silicone elastomer impregnated with 0.3 g of natural progesterone; Pfizer Animal Health, Hamilton, New Zealand) to synchronize oestrus in ewes. Three hundred twenty multiparous ewes of 2 native breeds (Naimi and Najdi) were equally and randomly allotted into 2 groups: Group A (FGA-30, n = 160) and Group B (CIDR®, n = 160). Both methods were inserted intravaginally for 14 days with intramuscular administration of 600 IU of eCG (Synchropart® Ceva Sante, Animale, France) at withdrawal time. Retention, vaginal discharge, and drawstring breakage rates were calculated at withdrawal time. The standing oestrous was detected using a vasectomized ram starting 24 h after progestagen withdrawal and repeated every 12 h (24 h, 36 h, 48 h, 60 h, and 72 h). The blood samples were collected at the time of progestagen withdrawal, after 24 h, and after 48 h (at time of AI). Follicular stimulating hormone, LH, oestradiol (E2), and progesterone (P4) serum concentrations were measured using commercial ELISA kits and micro-titrimetric plates. Laparoscopic insemination was performed 48 h after progestagen withdrawal. Pregnancy was diagnosed by ultrasonography at day 23 after insemination and confirmed at Day 35 and 60. Number of fetuses was recorded and confirmed at lambing. Pregnancy rate, fecundity percentage (litter size percentage), and prolificacy percentage (lambing rate) were calculated. The SAS programme was used for all analyses. Data were expressed in percentages except hormone levels, which were expressed as the mean ± standard error. Comparisons among groups were evaluated using Chi Square in all measured parameters except hormone levels, which were evaluated using an analysis of variance (ANOVA) test. A difference was considered significant at P < 0.05. The results revealed that retention rate was insignificantly different between the two groups. Drawstring breakage was observed only in FGA-30 and was absent in CIDR (9.33% v. 0). Moreover, vaginal discharge rate was significantly higher in FGA-30. Oestrus response was significantly higher in CIDR at 24 h and 48 h after progestagen withdrawal. Oestradiol and progesterone serum levels were significantly higher in CIDR, whereas LH and FSH serum levels showed insignificant differences. Pregnancy rate, twining rate, prolificacy, and fecundity were significantly higher in CIDR (75.71%, 33.96%, 1.34 and 1.01, respectively). These results show that although FGA and CIDR devices are efficient in synchronizing oestrus in ewes, CIDR provides higher oestrus response rate, pregnancy rate, twining rate, prolificacy, and fecundity. Consequently, the use of CIDR is recommended.
181 mtDNA COPY NUMBER IN OOCYTES OF DIFFERENT SIZES FROM INDIVIDUAL PRE- AND POST-PUBERTAL PIGS
Authors: Pedersen H. S. , Løvendahl P. , Larsen K. , Madsen L. B. , Callesen H.
Published: December 2014

Oocyte competence has been related to mtDNA copy number, but a large variation in mtDNA copy number between oocytes has been observed, caused by, e.g. oocyte donor and oocyte size (Sato et al. 2014 PLOS ONE 9, e94488; Cotterill et al. 2013 Mol. Hum. Reprod. 19, 444–450; El Shourbagy et al. 2006 Reproduction 131, 233–245). However, the correlation between size and mtDNA copy number in single oocytes has not been determined. This study describes the relation between oocytes of defined diameters from individual pre- and postpubertal pigs and mtDNA copy number. Cumulus-oocyte complexes were aspirated from ovaries of 10 pre- and 10 post-pubertal pigs. Cumulus cells were removed and the oocytes were measured (inside-ZP-diameter). Oocytes were transferred to DNAase-free tubes, snap-frozen, and stored at –80°C. The genes ND1 and COX1 were used to determine the mtDNA copy number. Plasmid preparations containing ND1 and COX1 were used to prepare standard curves. Q-PCR reactions using ND1and COX1 specific primers and probes were run in triplicate on a ViiA7 real-time PCR system using TaqMan (Applied BioSystems, Foster City, CA, USA). Standard curves showed high correlation coefficients (r2 = 0.99–1.00) and amplification efficiencies (COX1, 91–104%; ND1, 84–92%). As inter-assay control, standard curves were compared using interaction with dates, showing no differences. mtDNA copy number between groups was compared by ANOVA after log-transformation of data. Relationship between oocyte size and mtDNA copy number was analysed using linear regression. Data were analysed by SAS procedures, SGPLOT and GLM (SAS 9.3, SAS Institute Inc., Cary, NC, USA) with P < 0.05 as significance level. In total, 145 pre- and 93 post-pubertal oocytes were analysed (6–15 from each donor). Mean mtDNA copy number in oocytes from any individual donor was either high (≥100 000) or low (<100 000) with no differences between pre- and post-pubertal oocytes. No differences were detected in mtDNA copy number using either of the two primers (Table 1). No linear correlation was detected between oocyte size and mtDNA copy number in pre- and postpubertal oocytes (r2 = 0.00). The donor has a strong influence on oocyte mtDNA copy number in pre- and postpubertal pigs, which could influence individual fertility. mtDNA copy number does not seem to explain the higher developmental competence of neither post- compared to prepubertal pigs nor large compared to small prepubertal oocytes. The impact of individual on mtDNA copy number should be considered for experimental designs and future investigations.

Table 1.Analysis of pre- and post-pubertal oocytes
189 THE EFFECT OF VITAMIN D SUPPLEMENTATION ON ANTI-MULLERIAN HORMONE LEVELS IN REPRODUCTIVE-AGE WOMEN
Authors: Taheri M. , Modarres M. , Abdollahi A.
Published: December 2014

Vitamin D deficiency has been correlated with the infertility and lower clinical pregnancy following IVF. Anti-Mullerian hormone (AMH) plays a key role during follicle development; it has been recognised as a predictor of regular ovulation and probably IVF success. Considering the critical need for experimental human study to investigate the impact of vitamin D supplementation on ovulatory function, the aim of this study was to demonstrate the effectiveness of the vitamin D supplementation on AMH serum levels among reproductive-age women with vitamin D deficiency. 195 reproductive women (18–35 year-old) with confirmed vitamin D deficiency [serum 25(OH)D <75 nmol L–1] and without diagnosed polycystic ovary syndrome (PCOS) were enrolled to this controlled clinical trial. Participants were randomly assigned to a control group (n = 96) or an intervention group (n = 99). Women in the intervention group used 2000 IU day–1 vitamin D drops for 15 weeks. 19 participants were missed during the follow-up; finally the numbers of women in the intervention and control groups were 91 and 85, respectively. At the beginning of the study and after the intervention, 25-hydroxyvitamin D and AMH serum levels were quantified using enzyme immunoassay (EIA; Immunodiagnostic Systems, Boldon, UK) and ELISA (Beckman-Coulter Inc., Fullerton, CA, USA) methods respectively. The post-intervention AMH measurement was performed after 2–5 weeks in the same day-of-cycle on which basal AMH measurement was done. Paired t-test, independent t-test, and Pearson correlation were used as appropriate and a P-value of less than 0.05 was considered significant. Significantly low AMH levels were seen in the vitamin D deficient women of this study (14.46 ± 11.92 pmol L–1 in control group and 14.09 ± 11.52 pmol L–1 in intervention group). After the intake of vitamin D supplementation in intervention group, AMH levels were increased to 24.89 ± 12.47 pmol L–1, which were significantly different from the 15.43 ± 13.03 pmol L–1 in control group (P < 0.001). Correlation coefficients for AMH with pre-intervention and post-intervention vitamin D were r = 0.489 and r = 0.599 respectively (P < 0.001). Treatment of vitamin D deficiency increases AMH to the optimum levels. Vitamin D deficient women had low levels of AMH. These findings support other studies which found a correlation of poor IVF outcomes with low vitamin D levels. Vitamin D supplementation could be useful in the improvement of controlled ovarian hyper-stimulation/IVF outcomes in case of vitamin D deficiency.
190 EXPRESSION OF GENES ASSOCIATED WITH WINGLESS-RELATED MOUSE MAMMARY TUMOUR VIRUS SIGNALING IN THE PRE-IMPLANTATION BOVINE EMBRYO
Authors: Tribulo P. , Moss J. I. , Hansen P. J.
Published: December 2014

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔCT values, which were calculated by subtracting the CT value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the CT value of the sample. The relative transcript abundance was calculated as the 2ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development.
Research was supported by USDA-NIFA 2011-67015-30688.
194 EFFECT OF MATERNAL AGE ON THE GRANULOSA CELL TRANSCRIPTOME OF PREOVULATORY FOLLICLES IN CATTLE
Authors: Khan M. I. R. , Dias F. C. F. , Sirard M. A. , Adams G. P. , Singh J.
Published: December 2014

The objective was to determine how maternal age influences the transcriptome of the dominant follicle during the preovulatory period. We tested the hypotheses that delayed ovulation in aged cows is associated with 1) altered gene expression of granulosa cells of the preovulatory follicle and 2) decreased synthesis of progesterone by granulosa cells of the preovulatory follicle. Granulosa cells of preovulatory follicles were obtained 24 h after LH treatment from aged Hereford cows (17.0 ± 2.5 years; n = 6) and their daughters (9.0 ± 0.6 years; n = 6), and compared using bovine specific microarrays (EMBV3, EmbryoGENE, Québec, QC, Canada). Results were confirmed by RT-qPCR. A total of 1340 genes or gene isoforms were expressed differentially (≥2-fold change; P ≤ 0.05) in aged cows v. their younger daughters. Differentially expressed up- and down-regulated genes were related to 1) LH response (↑RGS2, ↑SERPINE2, ↑PTGS2), 2) cellular differentiation and luteinization (↑TNFAIP6, ↑GADD45B, ↓VNN1), and 3) progesterone synthesis (↑STAR, ↑HSD3B2, ↑NR5A2, ↑NR4A1). Intra-follicular concentration of progesterone was lower (P < 0.05) in aged v. young cows. Pathway analysis of the dataset revealed that mechanisms of delayed ovulation in aged cows may involve 1) post-receptor desensitization of G-coupled protein receptors, 2) inactivation of tissue plasminogen activator, and 3) delayed production of prostaglandin E2. In conclusion, transcriptome analysis of granulosa cells from aged cows revealed a delayed or suboptimal response to the preovulatory LH stimulus, represented by delayed cellular differentiation, luteinization, and progesterone synthesis.
This study was supported by grants from the National Science and Engineering Research Council of Canada, and the EmbryoGene Network, Canada. M.I.R. Khan was supported by graduate assistant scholarship from the Higher Education Commission of Pakistan.
Corrigendum to: 198 AGE EFFECT ON RELATIVE GENE EXPRESSION OF BOVINE SPERMATOGONIA
Authors: Giassetti M. I. , Moreira P. V. , Goissis M. D. , Oliveira de Barros F. R. , Delgado J. C. , Mendes C. M. , Assis P. M. , Assumpção M. E. O. d'Á. , Visintin J. A.
Published: December 2014

Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and animal transgenesis. However, the isolation of bovine SSCs is less efficient than for other species and it is lower for adult bulls. The goal of this study was to verify whether the relative gene expression of spermatogonia is affected by age after the laminin differential plating. Ten grams of parenchyma of testicles from pre-pubertal animals (5 months of age, n = 5) and bulls (3–4 years of age, n = 5) were minced and digested: colagenase (1 mg mL–1) for 30 min at 37°C and trypsin (2.5 mg mL–1) for 5 min at 37°C. Cells were plated (3 × 106 viable cells) in 60-mm culture dish covered with laminin (20 ng mL–1) and they were cultured for 15 min in high-humidity atmosphere with 5% of CO2 at 37°C. The adherent cells were recovered by enzymatic digestion with 0.1% trypsin for 1 min at 37°C. Viable cells were selected by the Trypan exclusion method, RNA was extracted from ~200.000 viable cells (Ilustra RNAspin Mini RNA, GE Healthcare, Waukesha, WI, USA) and cDNA synthesis was performed (SuperScript® VILO™, Invitrogen, Carlsbad, CA, USA). ITGA6 (integrin, α 6), SELP (Selectin P) and ICAM (Intercellular Adhesion Molecule 1) relative gene expression were determined by real-time RT-PCR (Mastercycler ep Realplex, Eppendorf International, Hamburg, Germany); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (actin β) were used as housekeeping genes. The statistical analysis was performed by QPCR_MIXED (SAS®, SAS Institute Inc., Cary, NC, USA). An α level at 0.05 was always adopted. ITGA6 and ICAM1 relative expression weren't effected by age; respectively, P = 0.2367 and P = 0.3583. However, SELP was highly expressed in adult bulls (P = 0.0022). Previously, ICAM1 and SELP have also been shown to mark aging haematopoietic stem cells and were more highly expressed in spermatogonia from adult mice than younger. However, SELP is expressed in more differentiated spermatogenic cells such as human sperm. The expression levels of ITGA6 did not seem to be significantly altered by age, indicating that age affects only certain SSC properties.
Financial support was provided by FAPESP (CEUA/FMVZ/USP 2509/2011).
218 DISTINCT EXPRESSION OF CALCIUM TRANSPORT CHANNELS, TRPV5, TRPV6, PMCA1, NCKX3, AND CaBP-9k IN THE DUODENUM, KIDNEY, AND PLACENTA DURING PREGNANCY
Authors: Yang H. , Ahn C. , Jeung E.-B.
Published: December 2014

Preeclampsia is a pregnancy-specific disease characterised by concurrent development of hypertension, proteinuria, and oxidative stress in the placenta. Preeclampsia-like genetic models were also developed by modification of preeclampsia-related genes, such as catechol-O-methyltranferase (COMT). In the current study, we hypothesised that inhibition of COMT alters the expression of calcium transport genes and contributes to induction of a preeclampsia-like condition, and the treatment regimen such as calcium supplementation ameliorates the imbalanced calcium metabolism and preeclamptic symptoms. In this study, we induced COMT inhibition in mice during pregnancy to reproduce physiological conditions associated with preeclampsia. The expression of genes known as hypoxia biomarker, HIF-1a, was highly induced in the placenta of this model. The overexpression of HIF-1a demonstrates that our experimental conditions closely were similar with preeclampsia. We measured the expression of several calcium transporters (TRPV5, TRPV6, PMCA1, and CaBP-9k) in the placenta, duodenum, and kidney after COMT inhibition on gestation day 17.5 (GD 17.5). Twenty-eight mice were divided into 4 groups [Vehicle, calcium, RO41–0960, and RO41–0960 and calcium (2% of calcium carbonate)]. In addition, we evaluated the calcium transporters in the kidney and duodenum of nonpregnant female mice. The placental TRPV5,6, PMCA1, and CaBP-9k were investigated by real-time RT-PCR. All experimental data was presented mean ± standard error of the mean (s.e.m.); P-values were calculated using one-way ANOVA. Placental TRPV5, TRPV6 and PMCA1 expressions were significantly down-regulated by COMT inhibitor (ro41–0960). In addition, the reduced PMCA1 expression in the placenta was reversed by calcium supplementation. Duodenal expressions of TRPV5, TRPV6, and PMCA1 were significantly decreased in the COMT-inhibited mice, and slightly recovered after calcium supplementation. Renal expression of TRPV5, TRPV6, and PMCA1 was also decreased by COMT inhibition, while it was reversed by calcium supplementation to the level of control. Duodenal- and renal calcium transporting genes, TRPV5, TPRV6, PMCA1, and CaBP-9k, were down-regulated by COMT treatment in female mice. Taken together, these results indicate that physiological changes observed in COMT inhibition showed similar symptoms as in preeclampsia, which may be related with disturbance of calcium metabolism during pregnancy.
226 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING CHEMICAL PACKETS THAT REGULATE CO2 AND O2
Authors: Saeki K. , Fujiki Y.
Published: December 2014

Bovine embryos are now routinely produced with oocytes collected from slaughterhouse ovaries or by transvaginal ovum pickup. The oocytes are matured, fertilized, and cultured in a water-jacketed CO2/O2 incubator. Gas phase in incubators is usually maintained at 5% CO2 in air for in vitro maturation (IVM) and IVF of oocytes and at 5% CO2, 5% O2, and 90% N2 for in vitro culture (IVC) of embryos. Here we investigated whether two chemical packets that regulate CO2 and O2 for culturing bacteria (Mitsubishi Gas Chemical, Tokyo, Japan) could be used to control the gas phase in vitro production (IVP) of cattle embryos. One packet (Anaero Pack-CO2) was maintained at a CO2 level of ~5% in a 2.5-L container and the other (Anaero Pack-MicroAnaero) was maintained at a CO2 level of 5–8% and an O2 level of 6 to 12%. Bovine cumulus-oocyte complexes (COC, n = 970) were collected from slaughterhouse ovaries, matured in HEPES-buffered TCM-199 (catalog number 12340–030, Invitrogen) supplemented with 10% FCS, 0.02 Armour unit mL–1 FSH and 1 µg mL–1 E2 for 22 h, and fertilized in medium IVF100 [Research Institute for the Functional Peptides Co. Ltd. (IFP), Yamagata, Japan] with frozen-thawed sperm (4 × 106 cells mL–1) for 6 h. Sperm and cumulus cells were removed from the oocytes. The denuded oocytes were cultured in IVD101 (IFP, 20 to 30 embryos/50 μL) for 8 days (Day 0 = IVF). Culture was carried out at 39°C with maximum humidity. Five different combinations of gas conditions were used for incubation (Table 1). Experiments were repeated 3 times. Cleavage and blastocyst rates were assessed on Day 8. Data were analysed by ANOVA followed by Fisher's PLSD test. In the five conditions, rates of matured oocytes (oocytes at MII, n = 210) were 70 to 73% and rates of normal fertilized oocytes (oocytes with 2 pronuclei, n = 310) were 67 to 75%. Cleavage rates of embryos after 8 days of culture (n = 450) were 68 to 75%, and rates of blastocysts from cleaved embryos were 25 to 40%. None of the above measures were significantly different among the 5 conditions (P > 0.05). These results indicate that gas phase control is not needed for IVM and IVF of bovine oocytes for their subsequent development. Anaero Pack-MicroAnaero (5–8% CO2, 6–12% O2) can be used for IVC of bovine embryos. The CO2-generating and deoxidizing packets can be successfully used to control the gas phase during bovine embryo production.

Table 1.Five different combinations of gas conditions used for incubation
230 ROLE OF PITUITARY HORMONES AND CUMULUS CELLS IN MODULATING THE DEVELOPMENTAL CAPACITY OF AGING BOVINE OOCYTES
Authors: Singina G. , Lebedeva I. , Taradajnic T. , Zinovieva N.
Published: December 2014

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).