bluuebunny/crossref_metadata_2025 huggingface dataset:
159 IN VITRO EMBRYO PRODUCTION FROM HOLSTEIN CALF OOCYTES RECOVERED BY LAPAROSCOPIC OVUM PICKUP
Authors: Baldassarre H. , Currin L. , Michalovic L. , Glanzner W. , Gutierrez K. , Bohrer R. , da Rosa P. , De Cesaro M. , Dicks N. , Schuermann Y. , Bordignon V.
Published: December 2015

Oocyte competence and reproductive biology in prepubertal heifer calves are not fully understood. Multiple publications have reported high oocyte yields recovered from calves aged 2–6 months old but low embryo development rates following in vitro embryo production. The objective of this study was to characterise the developmental competence of oocytes from young calves. We report herein the oocyte/embryo yields obtained from 6 Holstein heifer calves that were subjected to gonadotropin stimulation and laparoscopic ovum pick-up (LOPU) every 2 weeks, starting at 2 months of age and ending at 5 months of age. The LOPU was conducted under general anaesthesia with the animal lying in dorsal recumbency on a table with a 45-degree angle to facilitate the visualisation of reproductive organs. Briefly, looking through the laparoscope, the ovarian surface was exposed by pulling from the fimbria with an atraumatic grasping forceps. The follicle contents were aspirated using a 20 G needle mounted on a pipette, which was connected to a collection tube and a vacuum pump. Media and procedures for aspiration, in vitro maturation (IVM), IVF, and in vitro culture (IVC) were standard in use for commercial adult bovine embryo production. Because of the small number of animals and the multifactorial variables in play (age, number of previous treatments and aspirations, etc.), in this preliminary study we focused on the overall oocyte/embryo yield and the potential effects of LOPU on ovarian integrity. A total of 766 follicles were aspirated (avg. 17/calf per session) resulting in 625 cumulus-oocyte complexes (COC) recovered (avg. 14/calf per session; 82% recovery rate). A total of 457 (73%) COC were graded eligible for IVM, of which 353 cleaved (77%) and 109 (24%) reached a viable blastocyst stage at the end of IVC, of which 42 (38.5%) were graded as freezable. In balance, ~2 viable blastocysts/calf per session were produced. No adhesions or sequels were observed in the animals up to the last LOPU session, as well as 2 weeks after the last LOPU when the animals were evaluated by rectal palpation by an experienced OPU veterinary practitioner. Further studies will look into other aspects of oocyte developmental competence to better understand this biological process.
163 EFFECTS OF SERUM AND L-CARNITINE ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Zolini A. , Carrascal-Triana E. L. , Ruiz A. , Penitente-Filho J. M. , Hansen P. J. , Alves Torres C. A. , Block J.
Published: December 2015

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P < 0.05) development to the blastocyst (19.7 ± 1.1% v. 25.3 ± 1.4%) and advanced blastocyst (9.1 ± 0.8% v. 12.4 ± 1.2%) stages. While l-carnitine did not affect blastocyst development, advanced blastocyst development was reduced (P < 0.05) for l-carnitine at 3.03 mM (0 mM: 10.9 ± 1.2%, 0.75 mM: 12.2 ± 1.4%, 1.5 mM: 13.5 ± 1.5%, 3.03 mM: 7.0 ± 1.0%). Serum reduced (P < 0.01) re-expansion (78.1 ± 3.4% v. 65.5 ± 3.1%, 81.0 ± 3.0% v. 68.4 ± 2.7%, 78.4 ± 3.4% v. 65.8 ± 3.1%, for 24, 48, and 72 h, respectively) and hatching (52.0 ± 4.0% v. 39.8 ± 3.6%, 61.2 ± 4.1% v. 45.4 ± 3.8%, 61.2 ± 4.1% v. 45.4 ± 3.8%, for 24, 48, and 72 h, respectively) rates at all time points. In contrast, treatment of embryos with l-carnitine during culture increased (P < 0.05) post-thaw re-expansion rates at 24 and 48 h, regardless of concentration (Table 1). In conclusion, post-thaw viability of bovine embryos PIV can be improved by the addition of l-carnitine during culture. Moreover, the beneficial effects of l-carnitine on cryosurvival are not dependent on serum supplementation.

Table 1.Effect of addition of l-carnitine during culture on post-thaw re-expansion and hatching rates
170 IN VITRO EMBRYO PRODUCTION AND APOPTOSIS DETECTION USING BOVINE OOCYTES MATURED WITH IGF-I OR IGF-LongR3
Authors: De Chico P. L. M. , Recalde E. C. S. , Ikeda T. L. , Sudano M. J. , Landim-Alvarenga F.
Published: December 2015

The aim of this study was to compare the differential effects of the addition of IGF-LongR3 or insulin-like growth factor-1 (IGF-1), during in vitro maturation (IVM) of bovine oocytes on embryonic development, by analysing embryonic viability and apoptosis. Ovaries collected at a slaughterhouse were transported in saline solution (NaCl 0.9%) at 38°C. Follicles of 2–8 mm of diameter were aspirated. Only cumulus-oocyte complexes (COC) with homogeneous cytoplasm, surrounded by at least 3 layers of compact cumulus cells were selected in Dulbecco’ modified PBS 0.3% polyvinyl alcohol (PVA) and transferred to TCM HEPES at 38°C. Maturation medium was composed by TCM199, 0.2 mM pyruvate, 1 μg mL–1 FSH, 50 μg mL–1 LH, 100 μg mL–1 streptomycin sulfate, 100 UI mL–1 penicillin, and 85 μg mL–1 amikacin. Four experimental groups were determined using basic medium supplemented with fetal bovine serum (FBS; 10%), PVA (3 mg mL–1 Polyvinylpyrrolidone), IGF-1 (3.100 ng mL–1 insulin growth factor), LongR3-IGF (14.100 ng mL–1 long-chain human insulin growth factor 1-r3) respectively. The IVM was performed in Petri dishes (35 × 15 mm) with 90-μL droplets of the respective media, covered with sterile mineral oil, in 5% CO2 and 38.5°C temperature atmosphere for 22–24 h. The matured oocytes were fertilised with 2 × 106 spermatozoa mL–1 and incubated for 18 h. Embryos were denuded and cultivated in SOFaa medium (2.7 mM myoinositol, 0.2 mM pyruvate, 5 mg mL–1 BSA, 100 μg mL–1 streptomycin, 100 UI mL–1 penicillin, 85 μg mL–1 amikacin, and 25 μL mL–1 of FBS). Six repetitions were performed. After 7 days, blastocyst formation was analysed and all blastocysts were submitted to the TUNEL reaction. (In Situ Cell Death Detection Kit with Fluorescein, Roche®, Mannheim, BW, Germany), according to the technique adapted by Paula-Lopes and Hansen (2002).Green nuclei fluorescence (fluorescein isothiocyanate; FITC) was considered with fragmented DNA. Data were analysed by ANOVA), and least-squares means was used to verify differences of means using the PROC GLIMMIX model of SAS statistical software package (SAS Institute, Cary, NC, USA). Cleavage rate was similar for all groups. There was no statistical difference (P > 0.05) between groups regarding to embryo production. Most of the blastocysts obtained at Day 7 of culture had good quality (grade I). However, a difference (P = 0.03) on the number of expanded blastocyst was found between FBS (20.83 ± 3.22) and PVA (10.18 ± 3.22) groups. Furthermore, IGF-1 (12.03 ± 1.60) group showed a higher (P = 0.04, P = 0.02) apoptosis rates compared to groups FBS (7.96 ± 1.29) and PVA (6.97 ± 1.48). In the present study, it was observed that the addition of IGF-1 or LongR3-IGF during IVM provided a similar embryo production compared to FBS. On the other hand, embryos obtained from oocytes maturated on medium with the addition of IGF-1 had a high number of cells with fragmented DNA, indicating a more apoptosis.
181 IN VITRO FERTILIZATION (IVF) USING SEMI-DEFINED CULTURE CONDITIONS FROM LOW OR HIGH ANTRAL FOLLICLE COUNT PUBERTAL BEEF HEIFERS
Authors: Chase C. C. , Cushman R. A. , McNeel A. K. , Amundson O. L. , Perry G. A. , Tenley S. C. , Wood J. R. , Cupp A. S. , Vallet J. L. , Miles J. R.
Published: December 2015

To compare the in vitro fertilization (IVF) and production (IVP) of embryos from low and high antral follicle count (AFC) heifers, AFC were determined on 106 heifers using transrectal ultrasonography. Ten heifers with the lowest AFC (avg. 13.2) and 10 heifers with the highest AFC (avg. 27.4) with evidence of oestrous cyclicity were synchronised with 2 injections of prostaglandin F2α (PGF2α); half were harvested on Days 5 to 6 and half on Days 15 to 16 of the oestrous cycle. The IVF procedures included protocols for semi-defined media and were as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida, Gainesville, FL, USA). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilised using thawed frozen semen from a crossbred bull that was purified using Percoll gradient separation procedures. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in microdrops of development medium under oil, and cultured (5% CO2, 5% O2, balance N2, 38.5°C). On Days 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the mixed procedure of SAS (SAS Institute, Cary, NC, USA) and the model included the effects of collection day, group (high or low AFC), and their interaction. More COC (P < 0.0005) were collected from high than low AFC heifers (30.3 v. 9.3 ± 3.12 per heifer). Both the number and percentage of COC that cleaved had an interaction between collection day and group (P < 0.03). The interaction appeared to be due to low cleavage and development rates on 1 of 4 collection days (appeared not related to day of oestrous cycle). Although high compared to low AFC heifers had more COC that cleaved (18.7 v. 4.4 ± 1.84 per heifer), the percentage of cleaved COC did not differ (59.2 v. 49.8 ± 3.36%). There were no significant differences between high and low AFC heifers in the number of blastocysts (3.1 v. 0.6 ± 1.21 per heifer) or in the percentage of COC that developed to blastocysts (8.8 v. 5.2 ± 3.60%). In previous replicates (years), we reported that high AFC heifers had more COC collected, more COC that cleaved, and more COC that developed to blastocysts than low AFC heifers. In contrast, in this study numbers of COC that developed to blastocysts did not significantly differ between high and low AFC heifers. Additionally, the percentage of COC that cleaved, and that developed to blastocyst have been similar between high and low AFC heifers. Therefore, high compared to low AFC heifers may produce more IVP embryos; however, AFC does not appear to affect the competence of an oocyte to develop and mature to the blastocyst stage.
185 TRANSIENT RECEPTOR POTENTIAL CHANNEL-2 (TRPP2) REGULATES MOTILITY AND INTRACELLULAR CALCIUM OF PORCINE SPERM
Authors: Daigneault B. W. , Miller D. J.
Published: December 2015

Transient receptor potential channel-2 (TRPP2) is a membrane protein important for the regulation of calcium homeostasis in renal epithelial cells. Mutations in human TRPP2 cause enlarged cystic kidneys and contribute to polycystic kidney disease. In addition, TRPP2 functions have been described in some invertebrate sperm and are related to sperm-egg interactions and mating. Male Drosophila with mutated TRPP2 display a mild sperm motility phenotype but have a drastic reduction in fertility due to failed sperm migration and storage within the female tract. Although TRPP2 has critical roles for Drosophila sperm function, the protein has not been described in mammalian sperm. The TRPP2 mutations affecting sperm function could explain idiopathic subfertility that is not detected when evaluating sperm by routine analyses. Herein we report the location of TRPP2 in porcine sperm and have identified functions of TRPP2 in regulating sperm functions important for fertility. The TRPP2 was detected as a 110-kDa band in protein lysates from sperm after capacitation or mock incubation in conditions that do not capacitate sperm. With immunofluorescence, TRPP2 was most abundant on the head and principal piece of sperm with more consistent staining patterns when sperm were maintained in non-capacitating medium. Inhibition of TRPP2 by antiserum resulted in a reduction in sperm average path and curvilinear velocity and an increase in tail cross-beat frequency when sperm were incubated in capacitating conditions. Sperm incubated with TRPP2 antiserum also had a significant decrease in intracellular calcium concentration compared with control samples and failed to undergo an increase in calcium over 90 min that is characteristic of capacitating sperm. TRPP2 is a previously unreported mammalian sperm membrane protein that appears to function as an ion channel to regulate calcium and capacitation-like changes in porcine sperm.
This project is supported by Agriculture and Food Research Initiative Competitive Grant no. 2011–67015–20099 and 2015–67015–23228 from the USDA National Institute of Food and Agriculture.
189 CHARACTERIZATION OF THE FIRST SPERM-INDUCED CALCIUM TRANSIENT IN PIG OOCYTES
Authors: Wang C. , Machaty Z.
Published: December 2015

Fertilization in mammals is associated with repetitive elevations in the oocytes’ intracellular free calcium concentration. The elevations are triggered by the fertilizing sperm and are responsible for stimulating embryo development. In mouse oocytes, the sperm-induced calcium signal starts with a calcium rise that is larger and longer in duration than any succeeding transients. It also has unique characteristics: it begins with a rapid increase for 2–3 s followed by a shoulder, which is an inflection point that represents a brief decline in the rise of calcium levels. Once calcium level reaches its maximum, it decreases but remains elevated for several minutes while it is superimposed by several smaller calcium spikes. In bovine oocytes the situation is somewhat different. In this species, the first sperm-induced calcium transient is larger than the additional spikes but it lacks the sustained elevation phase and is not superimposed by small calcium rises. In the present study our purpose was to characterise the first sperm-induced calcium transient in pig oocytes. Oocytes were obtained from ovaries of prepubertal gilts collected at an abattoir and matured in vitro for 44 h. Mature oocytes were loaded with the calcium indicator dye fura-2; subsequently, they were either IVF or used for intracytoplasmic sperm injection (ICSI). Changes in their intracellular free calcium concentration were then immediately monitored using InCyt Im2, a dual-wavelength fluorescence imaging system. Characteristics of the first transients (including amplitude and duration) were compared to those of the additional ones using Student’s t-test. We found that in oocytes that underwent IVF (n = 11), the oscillations started 83.4 ± 23.2 min after adding the sperm to the oocytes. In the ICSI group (n = 10 oocytes) the calcium oscillations started sooner, 27.1 ± 17.7 min after injection. The average peak amplitude and the mean interval between the calcium transients varied among individual oocytes, but no significant differences were found between the IVF and ICSI groups (which on average were fluorescence ratio of 2.6 ± 1.1 and 23.5 ± 11.4 min, respectively; P > 0.1). The oscillation patterns showed slight differences between individual oocytes in terms of spike frequency, which has been described before and may be due to variations in the amount of sperm-derived activating factor present in the ooplasm. Most importantly, in all oocytes measured, the initial calcium spike showed no differences when compared to subsequent calcium transients: its amplitude and duration was similar to the additional transients. This points at potential species-specific differences in the regulation of calcium signalling in oocytes and provides essential information for the better understanding of the fertilization process.
This work was supported by Agriculture and Food Research Initiative Competitive Grant 2011–67015–30006 from the USDA National Institute of Food and Agriculture.
190 SPINDLIN 1 IS REQUIRED FOR METAPHASE II ARREST IN PORCINE OOCYTES
Authors: Choi J.-W. , Kim T. , Kim N.-H. , Cui X.-S.
Published: December 2015

Spindlin 1 (Spin1), is a histone methylation effecter protein. It can specifically recognise and bind to trimethylated histone 3 lysine 4 (H3K4). Spin1 localizes to the active rDNA repeats and facilitates the expressions of rRNA genes. For exploring the function of Spin1 in porcine oocyte and embryo development, siRNA of Spin1 (siSpin1) was used in the present study. Messenger RNA level of Spin1 was more highly expressed in the ovary than other tissues. During oocyte maturation, Spin1 was highly expressed from germinal vesicle to metaphase II stage in oocyes but sharply decreased after zygotic genome activation. The SPIN1 protein was localised to the cytoplasm and showed a similar expression pattern with mRNA through all stages of early embryonic development. Spin1 was successfully knocked down by siRNA injection. The siRNA of green fluorescent protein (siGFP) was used as positive control, and distilled water (DW) was used as a negative control. All were injected at metaphase II stage. Oocyte maturation was not altered by siSpin1 microinjection, but the rate of embryo development was significantly decreased from 4-cell stage when compared with the control group. The Spin1 had an effect on the blastocyst formation, which may have been altered by polymerase I-mediated rRNA gene transcription via regulation of H3K4 methylation. On the other hand, pronuclear formation was reduced by Spin1 siRNA. All data were analysed with a one-way analysis of variance, and differences between treatment groups were assessed by l.s.d. test, using SPSS software. In conclusion, Spin1 may be involved in metaphase arrest in oocyte and zygotic genome activation during early embryo development in pigs.
206 EFFECT OF ADDITION OF FOLLICULAR FLUID OR GROWTH DIFFERENTIATION FACTOR-9 ON IN VITRO MATURATION OF PORCINE OOCYTES DENUDED 20h AFTER THE START OF IN VITRO MATURATION
Authors: Ferré P. , Bui T. T. M. , Tran M. T. , Wakai T. , Funahashi H.
Published: December 2015

The interruption of communication between oocyte and cumulus cells (CC) can trigger meiotic resumption and exogenous additives, such as follicular fluid (FF) and growth differentiation factor-9 (GDF9), can improve oocyte quality and the developmental competence. This study was undertaken to examine if the absence and presence of FF from medium follicles (MF; 3–6 mm in diameter) or recombinant human GDF9 (Biovision, Milpitas, CA, USA) during the first or/and second half of in vitro maturation (IVM) had any effects on IVM of oocytes from small follicles (SF; 0.5–2 mm in diameter) or MF when the oocytes were denuded at 20 h after the start of IVM. Cumulus-oocyte complexes (COC) were aspirated from SF or MF of slaughtered prepubertal gilt ovaries. Groups of ~30 COC were cultured in a 300-μL drop of porcine oocyte medium containing 50 µM β-mercaptoethanol (mPOM) with or without 10% (v/v) FF and/or 100 ng mL–1 GDF9 at 39°C and 5% CO2 in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl c-AMP, 10 IU mL–1 eCG and 10 IU mL–1 hCG. After the first period of IVM, the CC surrounding the oocytes were removed and the denuded oocytes continued culture for IVM with or without FF or/and GDF9 in the absence of dibutyryl c-AMP and gonadotropins in the same medium for another 24 h. At the end of IVM, meiotic progression of the oocytes was examined by DAPI staining. Statistical analyses from at least 4 replicates data were performed by a 2-way ANOVA and a Tukey’s multiple comparisons test. Removal of CC 20 h after the start of IVM significantly improved the incidence of mature oocytes derived from SF (59.2–64.1% v. 41.6–43.1% in controls, P < 0.05) but not from MF (73.1–78.5% v. 70.6–71.8% in controls), whereas regardless of supplementation with FF or GDF9, the maturation rates were always significantly higher in the denuded oocytes from MF (72.4–83.6%) than SF (57.8–66.2%; P < 0.05). Despite of the origin of COC (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM were not affected by supplementation with FF or GDF9 during the first and/or second half of IVM (P > 0.05). In summary, CC removal from COC 20 h after the start of IVM promotes nuclear maturation of oocytes from SF. Exogenous additives such as GDF9 and follicular fluid from MF do not seem to affect the promotion of nuclear maturation in our experimental conditions.
210 POST-TRANSLATIONAL MODIFICATION OF HISTONE H4 ACETYLATION DURING IN VITRO MATURATION OF CANINE OOCYTES
Authors: Motheo T. F. , Arnold D. R. , Vicente W. R. R. , Macente B. I. , Filgueira F. G. F. , Chung D. G. , Almeida V. T. , Uscategui R. A. R. , Lopes F. L.
Published: December 2015

Proper in vitro oocyte maturation, crucial for in vitro fertilisation, is poorly understood and inefficient in dogs. Post-translational modifications of histones are involved in the meiotic process and preparation of oocytes for fertilisation. The present study aimed to evaluate post-translational histone H4 acetylation at lysine 12 (H4K12ac) and 16 (H4K16ac) in canine oocytes during in vitro maturation (IVM). Ovaries were retrieved from 1–7 year-old bitches undergoing routine ovariohysterectomy (OH). Grade I cumulus-oocyte complexes (COC) were recovered and evaluated before and after IVM in TCM 199 + 0.025 mM sodium pyruvate + 100 000 IU mL–1 penicillin G sodium + 100 mg mL–1 streptomycin sulfate, 0.003 g mL–1 BSA + 50 μL mL–1 Pluset® (500 UI FSH + 500 UI LH) + 1 μL mL–1 insulin-like growth factor (IGF) at 38.5°C in 5% CO2 for 72 h. After IVM, the nuclear stage of oocytes was established using Hoechst 33342 staining, and the acetylation patterns were investigated by indirect immunofluorescence staining of fixed immature and post-IVM oocytes (4% paraformaldehyde) with specific antibodies against the acetylated lysine residues, H4K12ac and H4K16ac. All stages were collected and evaluated simultaneously, and the experiment was repeated 4 times, with a total of 5–14 of oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIH ImageJ software 1.4, and data are presented as a percentage of the average fluorescence intensity of each specific antibody over the intensity of DNA, as determined by Hoescht staining. Immunofluorescence values were analysed using the least square ANOVA by the general linear model procedures of SAS (SAS Institute, Cary, NC, USA), and comparisons of means were further performed by Fisher’s Exact test. A probability level of P < 0.05 was considered significant. Our results indicate a variation in acetylation levels of H4K12 (P < 0.02) and H4K16 (P < 0.04) during meiosis. Fluorescence levels of H4K12ac decreased significantly when comparing immature (2.19 ± 0.62) with oocytes in metaphase I/II (MI/MII) (0.52 ± 0.14). Similarly, H4K16ac displayed a greater acetylation pattern in immature oocytes (1.20 ± 0.27) when compared to oocytes in germinal vesicle breakdown (GVBD; 0.58 ± 0.16) and MI/MII (0.62 ± 0.07) stages. These results support the hypothesis that, similar to other domestic species, post-translational modification of histone acetylation takes place during the meiotic process of oocyte IVM. Whether these histone modifications take place in a similar fashion during IVM remains to be investigated.
222 INCOMPLETE REPROGRAMMING OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PORCINE FETAL FIBROBLASTS
Authors: Choi K.-H. , Son D. , Lee D.-K. , Oh J.-N. , Kim S.-H. , Park T.-Y. , Lee C.-K.
Published: December 2015

Cellular reprogramming of committed cells into a pluripotent state can be accomplished by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. However, during reprogramming, it has been verified that failures of reactivating endogenous genes and epigenetic remodelling lead to partially reprogrammed cells exhibiting features similar to those of fully reprogrammed cells. In this study, partially reprogrammed induced pluripotent stem cells (pre-iPSC) were derived from porcine fetal fibroblasts via drug-inducible vector carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study aimed to investigate characteristics of pre-iPSC and reprogramming mechanisms. The pre-iPSC were stably maintained over an extended period having in vitro differentiation ability into 3 germ layers. The pluripotent state of pre-iPSC was regulated by modulation of culture condition. They showed naive- or primed-like pluripotent state in leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) supplemented culture conditions respectively. However, pre-iPSC could not be maintained without ectopic expression of transgenes. The cultured pre-iPSC expressed endogenous transcription factors (OCT4 and SOX2) except for NANOG known as gateway into complete reprogramming. In addition, endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occurred in female pre-iPSC. Given the results, we found that expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu for achieving faithful pluripotency in pig. Accordingly, investigating pre-iPSC could help us to improve and develop reprogramming methods via understanding reprogramming mechanisms in pig.
This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea.
225 AUTOLOGOUS TRANSPLANTATION OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE IN ANIMAL
Authors: Malik H. , Sharma V. , Saini S. , Guha S. , Malakar D.
Published: December 2015

The present study was carried out for isolation and culture of adipose tissue-derived mesenchymal stem cells of goat (gADSC) and dogs (1 dog was suffering from hip dysplasia and another dog from paraplegia) and their characterisation with different markers. Adipose tissue of goat and dog were aseptically isolated and treated with collagenase for 2 h in a CO2 incubator. The enzymatic digested cells were filtered through a 41-µm filter and cells were resuspended in cell culture flask containing medium DMEM/F12, 10% fetal bovine serum, and 50 μg mL–1 gentamycin. In vitro-cultured ADSC were characterised by amplification of mesenchymal stem cell (MSC)-specific surface marker genes of CD44, CD29, and CD166 in PCR and by immunocytochemistry of MSC-specific marker of CD44. For in vitro chondrogenesis, ADSC at passage 3 were incubated in DMEM/F12 containing 100 nM dexamethasone, 1.25 μg mL–1 BSA, and 10 ng mL–1 BMP-4 ITS (insulin-transferrin-selenium) for 3 wk. Chondrogenic differentiation cells were confirmed by Safranin O staining and positive expression of chondrocyte-specific marker genes Aggrecan: primers F-TTGGACTTTGGCAGAATACC and R-CTTCCACCAATGTCGTATCC, and Collagen II: primers F-AACCCTGGAACTGACGGAAT and R-CTCACCCGTTTGACCTTTCG in PCR. Dog ADSC-derived chondrocytes were aseptically injected at 1 × 106 cells kg–1 of BW into dogs with hip dysplasia and paraplegia. Both dogs recovered well after 1 month of autologous transplantation and were able to move freely. Then, 10 dogs having massive wounds were injected with heterologous undifferentiated mesenchymal stem cells at 1 × 106 cells kg–1 of BW and all dogs were cured in an average of 20 days. Then, the paralyzed and fractured dogs were further treated with undifferentiated MSC at 1 × 106 cells kg–1 of BW and most of the dogs were cured properly. These findings may have implications for defining the physiological roles of ADSC in arthritis, some orthopaedic problems, joint regeneration, and neurological disorders and several new applications leading to novel therapeutic opportunities.
230 EFFECT OF DIFFERENT CONCENTRATIONS OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ON EXPRESSION OF SELF-RENEWAL RELATED GENES IN GOAT (CAPRA HIRCUS) SPERMATOGONIAL STEM CELLS
Authors: Sharma A. , Shah S. M. , Saini N. , Singh M. K. , Singla S. K. , Palta P. , Manik R. S. , Chauhan M. S.
Published: December 2015

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily produced by Sertoli cells, is essential for the self-renewal of spermatogonial stem cells in vivo. The present study evaluated the effects of different concentrations of GDNF (human recombinant expressed in Escherichia coli) on expression of some self-renewal related genes in spermatogonial stem cells (SSC). The SSC were isolated from prepubertal goat testes (3–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin (DSA) lectin-coated dishes and Percoll density gradient centrifugation. The enriched cells were cultured on goat Sertoli cell feeder layer in DMEM + 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Primary SSC colonies were formed within 7 to 10 days. These colonies were characterised through alkaline phosphatase and immunofluorescence staining on Day 10 for different SSC-specific protein markers. Colonies were found to be positive for DBA, THY1, PLZF, UCHL1, OCT-4, SOX2, and NANOG, and negative for c-Kit expression. These colonies were cultured for 15 days without or with supplementation of GDNF forming following groups: (1) without GDNF (control), (2) 10 ng mL–1 GDNF, (3) 20 ng mL–1 GDNF, and (4) 40 ng mL–1 GDNF. RNA was isolated from 100 colonies from 3 different trials on Day 15 of culture, and relative expression of different self-renewal related genes was determined by qRT-PCR. Relative mRNA abundance of PLZF was higher (P < 0.05) following supplementation with 40 ng mL–1 GDNF than in other groups (i.e. control, 10 and 20 ng mL–1 GDNF). Expression of BCL6B and ID4 was found to be significantly higher (P < 0.05) after supplementation of GDNF at all concentrations compared with the control group. Expression of UCHL1 was higher with addition of 20 and 40 ng mL–1 GDNF (P < 0.05), whereas expression of THY1 was higher with supplementation of 10 ng mL–1 GDNF (P < 0.05). In conclusion, GDNF was found to benefit expression of goat SSC candidate genes at a concentration of 40 ng mL–1.
234 HORMONAL FOLLICLE STIMULATION IN HOLSTEIN COWS FOR IN VITRO EMBRYO PRODUCTION USING SPERM SORTED BY FLOW CYTOMETRY
Authors: Ferré L. , Bogliotti Y. , Chitwood J. , Kjelland M. , Ross P.
Published: December 2015

Transvaginal ultrasound needle-guided ovum pick-up (OPU) and in vitro embryo production (IVP) offer a reliable alternative to conventional embryo transfer to produce offspring. The success of OPU/IVP greatly depends on the number and quality of retrieved oocytes. The aim of this study was to compare OPU/IVP performance from stimulated Holstein cows. Holstein (Bos taurus) >8-year-old pluriparous open dry cows (n = 28) were used for OPU as oocyte donors. Follicular waves in all groups were synchronized by gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF), and CIDR administrated on Day 0, followed by stimulation treatments 48 h later. No pre-synch was used. Total hormone dosage were administrated as follows: Group 1: pFSH = 180 mg (Folltropin, Bioniche, Belleville, ON, Canada; n = 7), Group 2: pFSH/LH = 500 IU (Pluset, Calier, Barcelona, Spain; n = 7), Group 3: eCG = 1500 IU (eCG, Biogénesis-Bagó, Buenos Aires, Argentina; n = 7) and Group 4: Control (n = 7), no stimulation. All injections were performed intramuscularly (i.m.) twice a day, during three days. OPU was performed 48 (Group 1) or 24 h (Group 2 and 3) after the last injection. The control group received saline solution i.m. Follicles were classified according to diameter in 4 categories: small (2–5 mm); medium (6–9 mm); large (10–14 mm) and extra large (>15 mm). A Mindray DP-30 Vet (Mindray Medical, Shenzhen, China) was equipped with a micro-convex transducer 5.0- to 8.5-MHz probe along with a disposable 21G needle. The OPU flow rate was 15 mL min–1. Retrieved oocytes were classified according to IETS guidelines as viable (grade 1 + 2) and non-viable (grade 3 + 4). The IVP protocol was according to that in Reprod. Fertil. Devel. (2004, 16, 253). Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient (PureSperm, Nidacon, Mölndal, Sweden) and diluted to 1 × 106 sperm mL–1. ANOVA was used for comparisons of mean values and a chi-squared test was used for proportions. Results are presented in the Table 1. In conclusion, pFSH stimulation before ovum pick-up in Holstein cows increased the number of collected and viable oocytes, cleavage, embryo development, and hatching rates in comparison to other follicle stimulation hormones and non-stimulation. A cost-benefit analysis of these methods could be valuable in order to inform whether or not a stimulation protocol is necessary for a commercial IVP operation.

Table 1.Numbers of follicles, collected and viable oocytes, cleavage rate, blastocysts and hatching rate
237 EFFECT OF A PTEN INHIBITOR ON OVARIAN CONTENTS OF APOPTOSIS-RELATED PROTEINS AND ANTI-MÜLLERIAN HORMONE (AMH) IN A/J MICE
Authors: Suzuki O.
Published: December 2015

Strain/individual differences in superovulation efficiency with gonadotropins constitute a serious problem in mouse reproduction (Suzuki et al. 1996 Reprod. Fertil. Dev. 8, 975–980). Because PI3K/Akt signalling pathway is involved in ovarian folliculogenesis and phosphatase and tensin homologue deleted from chromosome 10 (PTEN) negatively regulates the process, I have been studying the effect of PTEN inhibitors on ovarian folliculogenesis and ovulation in mice to improve superovulation efficiency. In the present study, I compared ovarian contents of apoptosis-related proteins and anti-Müllerian hormone (AMH, an activity/number indicator of preantral and early-antral follicles) in A/J mice to examine the possibilities of an exogenous PTEN inhibitor to suppress follicular atresia and to promote folliculogenesis. At PMSG injections, 0 (control, n = 5) or 2 mg kg–1 B.W. (treated, n = 5) of a PTEN inhibitor, dipotassium bisperoxo (picolinato) oxovanadate (V), dissolved in Ringer solution were also injected to 28-day-old A/J female mice. Ovaries were collected 24 h after the injections. Ovarian protein contents of six proteins (shown in results) were measured by quantitative Western blots with β-actin as an internal control using whole ovary homogenates. Quantitativity of the Western blots were confirmed by calibration curves produced using a dilution series of sample mix (see Suzuki et al. 2011 Exp. Anim. 60, 193–196, for method details). Observed values were compared between control and treated groups by ANOVA. No significant difference in ovarian protein contents of apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9, cleaved PARP, BAX, and BCL-2) was found between the 2 groups (P = 0.951, 0.548, 0.095, 0.226, and 0.363, respectively; n = 5). On the other hand, ovarian AMH protein was more abundant in the treated group than that in the control group (P = 0.009). Although these results should be confirmed by more precise quantification methods such ELISA or RIA to eliminate possible lack of difference in apoptosis-related proteins due to methodology, they suggest that PTEN inhibitors can promote ovarian folliculogenesis but not suppress follicular atresia.
This work was supported by a grant from the Ministry of Health, Labour and Welfare of Japan.
238 PRODUCTION OF CLEAVAGE-RESISTANT PHYTASE TRANSGENIC PIGS BY HANDMADE CLONING
Authors: Zhang M. , Chen S. , Chen X. , Huang Y. , Wei L. , Chen B. , Wu Y. , Jiang Q. , Guo Y. , Wei Y. , Lin X. , Lan G. , Jiang H.
Published: December 2015

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions. Large quantities and high concentrations of manure waste that contains phytate phosphorus are generated. The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals. Enviropigs, producing phytase in the salivary glands and secreting the enzyme in the saliva, were first generated at the University of Guelph (Guelph, ON, Canada) in 1999. However, phytase is easily inactivated in digestive processing. To address this problem, we improved the transgene construct and successfully generated phytase transgenic pigs by handmade cloning. The Escherichia coli periplasmic phosphoanhydride phosphohydrolase (appA) gene was subcloned. Using fragment substitution method, we designed a phytase gene that was insensitive to cleavage by pepsin and trypsin and had a higher affinity for the substrate. After codon optimization, the designed phytase gene was named Cafp and subcloned downstream of the pig parotid secretory protein (PSP) gene promoter. The tissue-specific vector p-PSP-Intron-Cafp was constructed and transferred into Landrace fetal fibroblasts by electroporation. The cell lines carrying Cafp were used as nuclear donors in handmade cloning. Cloned embryos were cultured in vitro to blastocysts and transferred to recipient sows. The presence of Cafp was tested by PCR and sequencing of cloned pigs. Phytase activity in saliva, feed, and feces was detected by the ammonium molybdate method with a slight modification. Immunohistochemistry (IHC) was used to determine tissue-specific expression. Three cell lines carrying Cafp were obtained. We generated 1027 blastocysts; 712 were of good quality and transferred to 6 recipients. Fourteen piglets were born, of which 6 survived. The PCR and sequencing results showed that 7 (3 live and 4 dead) of the 14 piglets carried Cafp. Phytase activity in the saliva of the 6 live cloned pigs was tested at 4 months of age and only 1 pig had 0.155 FTU mL–1 enzyme activity. The enzyme in the other 2 pigs may be inactivated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, which represents a 25.4% decrease in fecal emissions compared with the average of controls. The IHC results on the 3 later dead, Cafp-positive pigs showed that the transgene was expressed only in parotids, confirming tissue-specific gene expression. In summary, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.
25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION
Authors: Whitworth K. M. , Murphy S. L. , Benne J. A. , Spate L. D. , Walters E. , Hickey R. , Nyberg S. L. , Wells K. D. , Prather R. S.
Published: December 2015

Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models.
This study was funded by the National Institutes of Health via U42 OD011140.
250 ANTI-MÜLLERIAN HORMONE: IMPLICATIONS FOR FOLLICULAR RESERVE, FOLLICULAR FUNCTION, AND FERTILITY IN THE MARE
Authors: Ball B. A. , Claes A. , Curry T. E. , Troedsson M. H. T. , Squires E. L. , Scoggin K. E.
Published: December 2015

Anti-Müllerian hormone (AMH) is a granulosa-cell derived glycoprotein which plays an important regulatory role during folliculogenesis. Antral follicle count (AFC) and AMH have been used in several species to assess ovarian reserve, but relatively little is known about these parameters in mares. The objectives of this study were to determine (i) plasma AMH concentrations and AFC in mares of different ages, as well as their repeatability within and across oestrous cycles; (ii) the relationship between plasma AMH concentrations and AFC with regard to age and follicle size; and (iii) endocrine and molecular changes in growing and preovulatory follicles in relation to AFC and peripheral AMH concentrations. In Exp. 1, young (3–8 years), middle-aged (9–18 years), and old (>18 years) mares were examined by transrectal ultrasonography over 2 to 3 oestrous cycles and changes in the number and size of all detectable antral follicles were recorded. Concentrations of AMH were determined by ELISA. In Exp. 2, molecular and endocrine differences in growing and dominant equine follicles were examined in relationship to variation in AFC, peripheral AMH concentrations and mare age. Follicular fluid oestradiol and AMH concentrations were determined by immunoassay, and expression of AMH, AMHR2, ESR1, ESR2, FSHR, IGF1, INHA, INHBA, and CYP19A1 was determined by quantitative RT-PCR. Antral follicle counts were significantly lower in old mares than in young and middle-aged mares, and AMH concentrations were significantly lower in old mares than in middle-aged mares. A positive relationship was detected between AFC and AMH, and this relationship varied by mare age with a strong correlation in older mares (ρ = 0.86; P < 0.0001), a moderate correlation in middle-aged mares (ρ = 0.60; P = 0.01), and no correlation (ρ = 0.40, P < 0.4) in young mares. Circulating AMH concentrations were significantly related to the number of antral follicles between 6 and 20 mm in diameter, and the repeatability of AFC and AMH concentrations was high within and between oestrous cycles. Within growing follicles, expression of AMH, AMHR2, ESR2, and INHA in granulosa cells was significantly associated with AFC and plasma AMH concentrations, whereas expression of ESR1 and FSHR were significantly correlated with AMH concentrations alone. In follicular fluid, AMH concentrations declined and oestradiol concentrations increased in dominant compared to growing follicles. Our findings indicate that the relationship between AMH and AFC varies across age groups, and that variations in AFC and plasma AMH concentrations are associated with molecular differences in the equine follicle, which, in turn, may have an impact on follicular function.
Funded by the Albert G. Clay Endowment at the University of Kentucky.
256 GENOMIC EVALUATION OF FERTILITY TRAITS AND DISCOVERY OF HAPLOTYPES THAT AFFECT FERTILITY OF US DAIRY CATTLE
Authors: Wiggans G. R. , Null D. J. , Cole J. B. , Norman H. D.
Published: December 2015

Genomic evaluations of dairy cattle became official in the United States in January 2009 for Holsteins and Jerseys, and later for Brown Swiss, Ayrshires, and Guernseys. Up to 33 yield, fitness, calving, and conformation traits are evaluated, and the fertility traits included daughter pregnancy rate and heifer and cow conception rates. Additional fertility traits, such as age at first calving and days from calving to first insemination, also are being studied. Male fertility (sire conception rate) is evaluated phenotypically rather than through genomics. Over 1 million animals have genotypes in the national database, which reflects collaboration with Canada and Europe. Most of the genotypes are from females and are from genotyping chips with <30 000 single nucleotide polymorphisms (SNP). To combine data across chips, genotypes are imputed to a set of >77 000 SNP. The imputation process involves dividing the chromosome into segments of approximately equal length and determining the paternal or maternal origin of the alleles. Because some segments were never homozygous, they were assumed to contain an abnormality that resulted in early embryonic death. If a decrease in sire conception rate could be associated with a bull that was a carrier of such a chromosomal segment, the haplotype was designated as affecting fertility. Once the region was identified, bioinformatic analysis was used to discover the causative variant for many of those haplotypes. Accuracy of genomic evaluations is determined by size of the reference population and heritability of the trait. The reference population for Holsteins includes >180 000 bulls and cows. Because fertility traits have low heritabilities, genomic information is particularly useful in improving evaluation accuracy. Accuracy of fertility evaluations is expected to increase further by discovering causative variants for various aspects of conception and gestation through investigation of sequence data.