bluuebunny/crossref_metadata_2025 huggingface dataset:
183 IN VITRO EMBRYO PRODUCTION: A TOOL TO PRESERVE THE THREATENED WOOD BISON (BISON BISON ATHABASCAE)
Authors: Cervantes M. P. , Palomino J. M. , Anzar M. , Mapletoft R. J. , Mastromonaco G. , Adams G. P.
Published: December 2015

In vitro embryo production is being developed as a tool to restore genetic diversity and eliminate endemic disease in wood bison. In a recent study in wood bison, we found that more oocytes reached maturity after 30 h v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). An additional 4 h of in vitro maturation after an in vivo maturation period of 30 h also had a positive effect on developmental competence. The present study was designed to test the hypothesis that extending the in vivo maturation time (i.e. extending the interval between hCG treatment and cumulus-oocyte complex (COC) collection) from 30 to 34 h will improve in vitro embryo production in wood bison. Follicular wave development was synchronised among female wood bison (n = 28, 6 to 10 years old) by transvaginal follicular ablation. The study was done in 4 replicates (n = 7 bison per replicate). Bison were given FSH 1 day (300 mg) and 3 days (100 mg) after ablation for ovarian superstimulation, and hCG (2500 IU) 5 days after ablation to induce COC maturation in vivo. Bison were divided randomly into 2 groups (n = 14/group) in which COC were collected transvaginally at either 30 h or 34 h after hCG treatment. Expanded COC from the 30 h group were fertilised after 4 h of in vitro maturation, while expanded COC from the 34 h group were fertilised immediately. Oocytes and sperm were co-incubated (Day 0 = day of fertilization) for 18 h at 38.5°C in 5% CO2 in air and high humidity. Presumptive zygotes were cultured in 4-well dishes containing 500 μL well–1 of CR1aa medium at 38.5°C, 5% CO2, 5% O2, 90% N2 and high humidity, and assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). Data were compared between groups by Chi-squared analysis. No effect of replicate was found. Compared to the 30 h group, the 34 h group had a greater cleavage rate [55/74 (74%) v. 49/86 (57%); (P < 0.05)], and a greater blastocyst rate on Day 7 [25/74 (34%) v. 9/86 (10%); (P < 0.05)] and Day 8 [(40/74 (54.1%) v. 32/86 (37.2%); (P < 0.05)]. We concluded that an extended period of in vivo maturation is beneficial for embryo production after in vitro fertilization in wood bison.
We thank Vetoquinol Canada for providing FSH (Folltropin-V) and hyaluronan (MAP-5) and thank Merck Animal Health for hCG (Chorulon).
195 HEAT STRESS ALTERS THE TRANSCRIPTOME OF MATURING BOVINE OOCYTES
Authors: Rispoli L. A. , Payton R. R. , Gondro C. , Saxton A. M. , Edwards J. L.
Published: December 2015

Direct exposure of maturing oocytes to a physiologically relevant elevated temperature reduces embryo development after fertilisation and has been coincident with reduced de novo protein synthesis. Mechanisms responsible for heat-induced reductions in protein synthesis are unknown but may be related to alterations in the transcriptome of the maturing oocyte. To determine the extent to which this may occur, the impact of heat stress on the maternal pool of RNA in bovine oocytes was assessed using microarrays. After maturation for 24 h at 38.5°C (control) or 41°C (first 12 h only, 38.5°C thereafter; heat stress) oocytes were denuded from associated cumulus cells and lysed for RNA extraction or underwent IVF to assess developmental competence. Total RNA from oocytes was amplified by 3′-poly(A) priming or a combination of 3′-poly(A) and internal priming because oocyte transcripts may or may not have a polyadenylated tail. Amplified RNA was hybridised to GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA, USA; 8 oocyte pools per treatment were collected on 7 different occasions and amplified by 2 methods; n = 32 chips). Differential transcript abundance was determined using R and Bioconductor with only probes having a P < 0.01, a fold change of at least 1.3, and called present for at least half the arrays. Functional annotation of selected transcripts was performed using Gene Ontology and KEGG annotations (Bos taurus build 4.0) and DAVID (v 6.7) with significance level set at P < 0.10. Coincident with reduced blastocyst development (28.3 v. 15.2% for control v. heat stress, respectively; SEM = 3.6; P < 0.0003), heat stress altered the abundance of 159 transcripts (22 increased, 137 decreased); 130 of these were annotated. Use of DAVID demonstrated enrichment of genes important for mitochondrial function and RNA processing. Towards validating certain findings, the relative abundance of 3 mitochondrial transcripts (NDUFC2, COQ3, ATP5O) were assessed by quantitative PCR on non-amplified RNA from the oocyte samples used for the microarray study. Gene-specific primers were designed for 5′ and 3′ ends of transcripts when possible. Exposure to elevated temperature during the first 12 h of oocyte maturation reduced transcript levels of NDUFC2 at the 5′ and 3′ ends (P < 0.0001 and P = 0.003), COQ3 at the 3′ end (P = 0.02) and ATP5O at the 5′ end (P = 0.02). In conclusion, exposure of maturing cumulus-oocyte complexes to a physiologically-relevant elevated temperature altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function.
This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.
204 INADEQUACY OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN CULTURE MEDIUM REDUCES THE VIABILITY OF CUMULUS CELLS AND PREVENTS IN VITRO MATURATION OF PORCINE OOCYTES
Authors: Bui T. T. M. , Ferré P. P. , Tran M. T. , Wakai T. , Funahashi H.
Published: December 2015

Recently, vascular endothelial growth factor (VEGF) has been regarded as an important factor associated with not only follicle development but also meiotic competence of oocytes. However, the mechanism of how VEGF works is poorly understood. In this study, we investigated in vitro maturation (IVM) of oocytes from different sizes in the absence or presence of a VEGF receptor inhibitor, Axitinib. Cumulus-oocyte complexes (COC) were obtained from small follicles (SF; l < 3 mm in diameter) and medium follicles (MF; 3–6 mm in diameter). Each group of 30–40 COC with at least 3 layers of clear and compact cumulus cells (CC) was cultured in 500 μL of modified porcine oocyte medium (POM-β-mercaptoethanol) supplemented with 10 IU mL–1 eCG, 10 IU mL–1 hCG and 1mM dibutyryl-cyclic-adenosine monophosphate (dbc-AMP) for the first 20 h and then without those supplements for another 24 h at 39°C, 5% CO2 in air. During the first 20 h of IVM, culture medium was also supplemented with or without 1.25 nM Axitinib. At 20 h and 44 h after the start of IVM, the oocytes were denuded and stained with 4′6-diamidino-2-phenylindole (DAPI) to observe the nuclear stages. At 20 h after the start of IVM, some COC were also stained with PI and SYBR Green I to evaluate the ratio of live/dead cumulus cells. Statistical analyses of data from 5 replications were analysed by ANOVA and Tukey’s multiple comparison test. As compared with controls at 20 h after the start of IVM, the number of dead cumulus cells increased significantly in the groups treated with Axitinib, regardless of COC derived from MF and SF (16.8 v. 43.1% in MF and 25.3 v. 57.7% in SF, P < 0.01, respectively). At that time, a majority of oocytes from MF and SF remained at the germinal vesicle (GV) stage in controls (89.8 and 84.6%, respectively), but the percentage significantly reduced in the presence of Axitinib (57.9 and 48.9% of oocytes from MF and SF, respectively) and proceeded around the metaphase-I stage (37.5 and 44.8% of oocytes from MF and SF, respectively). At 44 h after the start of IVM, lower maturation rates were observed in oocytes treated with Axitinib than controls (35.0 v. 81.2% in MF; 20.1 v. 49.0% in SF; P < 0.01). In conclusions, VEGF plays an important role in maitaining the viability of cumulus cells. The presence of VEGFR inhibitor caused the oocytes to develop uncontrollably, even in the presence of dbc-AMP. Moreover, the deficiency of VEGF prevented oocytes fully competent to resume meiosis and arrest to metaphase II.
208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT
Authors: Yoon J. D. , Lee E. , Hyun S.-H.
Published: December 2015

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.
211 PRODUCTION OF PROGESTERONE FROM CANINE CUMULUS CELLS MATURED IN VITRO WITH STEROIDS AND GONADOTROPIN
Authors: Apparicio M. , Covizzi G. J. , Alves A. E. , Pires-Butler E. A. , Motheo T. F. , Macente B. I. , Nogueira G. P. , Vicente W. R. R.
Published: December 2015

The canine reproductive physiology is distinct from other mammals, especially concerning the role of progesterone in ovulation, oocyte maturation, and fertilisation. In vitro maturation of canine oocytes is challenging mostly due to the culture conditions required for this species and the lack of knowledge about the factors involved in meiotic resumption and complete maturation. To obtain more information about this physiological phenomenon, the present study was designed to investigate the ability of canine cumulus cells to secrete progesterone in the maturation medium during in vitro culture for up to 96 h. Ovaries were collected from 18 bitches at different reproductive status (follicular, luteal, and anestrous stages; various breeds, age 1–7 years) by routine ovariohysterectomy and sliced in PBS-polyvinyl alcohol (PVA) to release the cumulus‐oocyte complexes (COC). A total of 896 COC grade I (2 or more dense layers of cumulus cells and darkly pigmented cytoplasm) were selected for culture. Oocytes were randomly allocated in 2 media consisting of TCM-199 (Sigma Chemical Co., St. Louis, MO, USA) with antibiotics, 10% FBS, 2.2 mg mL–1 sodium bicarbonate, and 20 μL mL–1 pyruvic acid (control) and control + hormones (25 IU mL–1 hCG (Sigma), 1 mg mL–1 progesterone (Sigma), 1 mg mL–1 estradiol (E2; Sigma). The COC were incubated at 38°C, 5% CO2 in air and cultured for 24, 48, 72, and 96 h. Maturation medium for in vitro culture of COC was collected at 0, 24, 48, 72, and 96 h and checked for progesterone content by radioimmunoassay (RIA). Maturation rates (meiotic configuration) were also evaluated at each culture period by using Hoechst 33342 (Sigma). Polynomial regression analysis was applied to assess variation of progesterone concentration in the maturation medium during the 96 h of IVM. Progesterone was detected in the control and hormonal media; however, in the control medium the concentration was <0.5 ng mL–1. In the hormonal medium, there was a decrease in progesterone concentration at 24 h in all groups (ranged from 461.8 to 587.2 ng mL–1) followed by a progressive increase until 72 h in follicular group (1371.4 ng mL–1) and until 96 h in luteal and anestrous groups (934.4 and 669.4 ng mL–1, respectively). Maturation rates (metaphase II oocytes) were higher at 72 h and decreased at 96 h in all groups. The results of this study suggest that hormonal supplementation is necessary for canine oocyte to resume meiosis and to cumulus cells be capable of producing progesterone. Moreover, progesterone secretion by cumulus cells seems to be related to the reproductive status of the donor bitch once higher concentration of progesterone was detected in medium from follicular group and lower in anestrous group.
215 PRODUCTION OF PROGENY WITH SEXED WHITE HANWOO (KOREAN NATIVE ALBINO CATTLE) SEMEN
Authors: Choi J. S. , Choe C. Y. , Kim D. K. , Seong H. H. , Kim S. W.
Published: December 2015

The aim of the study was to produce sexed progeny of the genetically confined White Hanwoo (albinism). A White Hanwoo bull was selected as the semen donor. The X-bearing sperm were sorted by MoFlo XDP cell sorter from Korea Sexing Biotech (Daegu, South Korea). To compare pregnancy and birth rates as well as semen quality, a Korean proven bull (KPN) was used as control. Total number of nonsorted sperm was 20 × 106 per straw. Double insemination of 2 or 4 × 106 sexed and frozen X-bearing sperm were performed on Hanwoo heifers. The abnormality of nonsorted albino spermatozoa (5 ejaculates) was 24.9 ± 7.31% (but 18.7 ± 6.2% C in KPN 768, derived from 5 straws) with a higher number of distal reflex abnormality in midpiece (11.7% compared with that of KPN 768, 5.6%). There were no differences in pregnancy and birth rates when 2 or 4 × 106 X-bearing sperm were inseminated but nonsorted semen had higher pregnancy rates (P < 0.05). The pregnancy rates of KPN 768, 2 × 106 cells, and 4 × 106 cells X-bearing sperm of White Hanwoo cattle were 85.0% (17/20), 26.3% (5/19), and 50% (7/14), respectively. The birth rates were 80.0% (16/20), 15.8% (3/19), and 21.4% (3/14), respectively. The female offspring rates were 43.8% (7/16), 100% (3/3), and 100% (3/3) (P < 0.05). These results indicated that sex-sorted albino Hanwoo semen could be used for the production of wanted progeny with 2 × 106 cells/straw for AI to heifers.
22 PRODUCTION OF PORCINE TRANSGENIC CELL LINE INSERTED WITH SV40LT, EGFRvIII Gene, AND INDUCIBLE CreERT SYSTEM
Authors: Hwang S. U. , Yoon J. D. , Eun K. Y. , Kim H. G. , Hyun S. H.
Published: December 2015

Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P < 0.05) and blastocyst formation rates (19.5 ± 1.2 and 5.6 ± 1.8 in the normal and SV40LT group, respectively; P < 0.05) of transgenic SCNT embryos was significantly lower than the case of using normal cells. In addition, we evaluated the transgenic SCNT embryo development of the donor cell transfected with SV40LT and HrasV12 genes (SV40LT+HrasV12 group). As a results, there was no significant difference between the groups in the cleavage rates, but blastocyst formation rates of transfected SCNT embryos (SV40LT+HrasV12 group) was significantly lower than the case of using normal cells (19.5 ± 1.2 and 6.2 ± 1.8 in the normal and SV40LT+HrasV12 group, respectively; P < 0.05). Genes SV40LT or HrasV12 showed a negative effect on SCNT cloned embryo development. Therefore, a Cre/loxP inducible system was applied to producing donor cells transfected with EGFRvIII and SV40LT gene. As a result, the cleavage rates (73.8 ± 4.0 and 68.6 ± 6.6 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) and blastocyst formation rate (19.5 ± 1.2 and 23.0 ± 3.7 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) were improved to the same level, when used as a donor cell to a normal cell. In conclusion, these results indicated that harmful effects of transgenic SCNT embryo development caused by the characteristics of the inserted genes can be overcome through the inducible system. Further studies are needed to experiment with mRNA expression of apoptotic gene and target gene in 4- to 8-cell embryos and blastocysts.
This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.
221 DERIVATION OF INSULIN-PRODUCING CELLS FROM HUMAN DERMAL FIBROBLASTS AT INTERMEDIATE PHASE OF REPROGRAMMING
Authors: Kang J. H. , Park S. M. , Heo S. Y. , Kang J. , Lee S. Y. , Ahn K. S. , Lee S. G. , Shim H.
Published: December 2015

Process of somatic cell reprogramming into induced pluripotent stem (iPS) cells can broadly be divided into an early stochastic phase and a later, hierarchical and more deterministic phase of gene activation. Between the stochastic onset and the deterministic progression while approaching pluripotency, cells upon reprogramming undergo a transient activation or repression of developmental regulators. In these intermediate cells yet acquiring pluripotency, many somatic gene expressions are lost, while various lineage-specific factors are activated. In the present study, we produced pancreatic β cells by exposing human dermal fibroblasts (HDF) at the intermediate phase of reprogramming to pancreas-specific culture conditions. To obtain the intermediate cells, reprogramming factor Oct4, Sox2, Klf4, and c-Myc were transduced into HDF using retroviruses. By Day 9 after transduction, the expression of fibroblast-associated Thy1 was lost, while the early reprogramming marker CD49d was activated, suggesting that the cells are at the early stage of intermediate phase. These cells were capable of differentiating into 3 germ layers when cultured in tri-differentiation medium (including 100 ng mL–1 Activin A, 0.1 ng mL–1 Wnt3 for endodermal differentiation; 20 ng mL–1 BMP4, 20 ng mL–1 Activin A for mesodermal differentiation; 10 μM SB431542, 600 ng mL–1 Dorsomorphin, and 35 ng mL–1 Noggin for ectodermal differentiation) and expressing lineage-specific markers Sox17 (endoderm), Brachyury (mesoderm), and Otx2 (ectoderm). Further, the endodermal cells derived from HDF at intermediate phase of reprogramming were cultured in pancreas-induction medium (KO DMEM with 1% insulin-transferrin-selenium (ITS), 10 ng mL–1 BMP4, 10 mM nicotinamide, 10 μM forskolin, 10 μM dexamethasone, and 50 ng mL–1 exendin-4). After 5 days in culture, cells expressed pancreatic progenitor marker Pdx1, as examined by immunostaining and fluorescence-activated cell sorting (FACS) analysis. After culture for additional 10 days, the cells formed spherical clusters and expressed pancreatic islet-specific genes including insulin, somatostatin, glucagon, Nkx6.1, and MafA, as determined by RT-PCR. Moreover, the production of insulin and C-peptide from the derived β cells was confirmed by immunocytochemistry and FACS analysis. In response to glucose stimulation, these cells secreted insulin and C-peptide, as measured by ELISA. The present study demonstrates that fully differentiated functional cells could be converted from somatic cells at intermediate phase of reprogramming by exposing cells to specific culture conditions. The results shown in this study may provide a faster and potentially safer way to produce functional pancreatic cells without isolation and differentiation of iPS cells.
235 ADMINISTRATION OF LEVOTHYROXINE IMPROVES THE PROPORTION OF TRANSFERABLE EMBRYOS FOLLOWING SUPEROVULATION IN JAPANESE BLACK CATTLE
Authors: Tominaga N. , Tomomatsu M. , Nakamura M. , Ideta A. , Konishi M. , Aoyagi Y.
Published: December 2015

The thyroid gland has a major impact on the human female reproductive system. However, its influence on reproductive competence in livestock is unclear. In cattle, superovulatory treatments often lead to high numbers of ova/embryos but low numbers of transferable embryos. Levothyroxine is a synthetic thyroid hormone that is chemically identical to thyroxine (T4), and binds to T4 receptors found in follicle cells. We aimed to determine whether levothyroxine treatment (LT) could improve the proportion of transferable embryos after superovulation in Japanese black cattle (Wagyu). First, we observed associations between the concentrations of T4, free triiodothyronine and thyroxine, and superovulatory responses (n = 95). Blood was collected before superovulatory treatment by jugular venipuncture, and serum was separated and stored at –20°C until being assayed for thyroid hormones. Second, Wagyu cattle showing a low proportion of transferable embryos (<30%) were assigned randomly to an LT group (n = 10) or a control group (no LT, n = 15); the LT was administered by 3 i.m. injections of 2000 mg of levothyroxine given 3 days apart; Controls received no injection. On the last day of LT, hormonal stimulation for superovulation was started as described (Ideta et al. 2007 Cloning Stem Cells 9, 571–580). The proportion of transferable embryos was compared with the same individual before/after LT. Data were analysed using Student’s t-tests. There were no significant associations between the concentrations of thyroid hormones and the superovulatory responses (no. collected oocytes/embryos, or the proportions of transferable embryos or unfertilized oocytes). However, LT dramatically increased the proportion of transferable embryos in these low-responding Wagyu cattle (Table 1). In conclusion, administration of levothyroxine improved the proportion of transferable embryos following superovulatory treatment. Further studies will be needed to investigate how LT improves the quality of bovine embryos.

Table 1.Effect of levothyroxine treatment on the proportion of transferable embryos after superovulation in Wagyu cattle (means ± SE)
254 SYSTEMS BIOLOGY OF SPERM AND BULL FERTILITY
Authors: Memili E.
Published: December 2015

Bull fertility, the ability of the spermatozoon to fertilize and activate the egg as well as support early embryonic development, is crucial for efficient production of cattle. With the major advances in fundamental science of reproduction, such as genomics, and in biotechnology, such as assisted reproductive technologies, bull influence on herd production has been accentuated. In addition to providing half of the genome for the zygote, sperm also contribute proteins, transcripts, and epigenetic traits that play important roles in sperm physiology, fertilization, and early embryonic development. Recently, using sperm from Holstein bulls with different in vivo fertility data, we have demonstrated that sperm Protamine 1 protein and a panel of microRNA are associated with bull fertility. In addition, molecular and cellular biology experiments on the analyses of sperm nuclear proteins showed that bull sperm have significant levels of histones that are also associated with bull fertility. This presentation will convey the results of most relevant research on discovery of sperm biomolecular markers associated with semen quality and bull fertility. Because of the available field data in bull fertility and the physiological similarities between bovine and other mammals, research results stemming from the systems biology of bull fertility are applicable to other species as well.
38 QUAIL EGG YOLK IN CITRATE EXTENDER IS SUITABLE FOR CRYOPRESERVATION OF NGUNI BULL SEMEN
Authors: Seshoka M. M. , Mphaphathi M. L. , Mafolo K. S. , Nkadimeng M. , Raphalalani Z. C. , Kanuya N. L. , Nedambale T. L.
Published: December 2015

Traditionally, commercial hen egg yolk has been used in extenders or freezing media because of its easy availability. However, the use of quail egg yolk has not been used for preserving Nguni bull semen. The aim of the study was to compare the suitability of different quail egg yolk concentrations (5, 10, 15, and 20%) for cryopreserving Nguni bull semen. Semen was collected from 14 stud Nguni bulls with the aid of electro ejaculator. Collected semen samples were kept in a thermos-flask containing warm water at a temperature of 37°C and transported to the laboratory for further analyses. The sperm motility traits were evaluated using computer-aided sperm analysis prior extension. Semen samples were then randomly allocated into 5, 10, 15, and 20% of quail egg yolk and 20% concentration of chicken egg yolk (control) in citrate extender. The extender was supplemented with 12% of glycerol (Seshoka et al. 2012) as a cryoprotectant, and semen samples were diluted (1 : 2) and equilibrated for a period of 4 h at 5°C. After equilibration, semen samples were loaded into 0.25-mL straws, placed into a controlled rate programmable freezer, and stored in a LN tank (–196°C) until thawing. Frozen semen straws were thawed in a water bath at 37°C for 1 min. Thawed semen was evaluated for sperm motility traits using a computer-aided sperm analysis system. Data were analysed with ANOVA. A significant difference was recorded between fresh total sperm motility rate (99%) and frozen-thawed semen samples with either 5% (87.3%) quail or 20% (87.6%) chicken egg yolk citrate extender compared with 10% (92.6%), 15% (91.2%), or 20% (89.9%) quail egg yolk citrate extender. Moreover, fresh semen also resulted in a significantly higher progressive sperm motility rate (39.3%) as compared with frozen-thawed with 5% (26.2%) or 20% quail (28.5%) or 20% chicken (22.7%) egg yolk citrate extender. The results also demonstrated that the use of 10, 15, and 20% quail egg yolk in citrate extender yielded comparable results on total sperm motility with fresh semen as compared with 5% quail and 20% chicken egg yolk. In conclusion, quail egg yolk extender provided sufficient cryo-effectiveness to cryopreservation of Nguni bull semen.
42 DIFFUSION OF THE CRYOPROTECTANTS ETHYLENE GLYCOL AND GLYCEROL INTO DAY 9 TO 11 EQUINE EMBRYOS: PRELIMINARY RESULTS
Authors: Chenier T. , Foster B. , Gonzalez L. , Schlaf M.
Published: December 2015

The impermeability of the capsule to cryoprotectants (CPA) has been blamed for poor cryosurvival of equine embryos. We hypothesised that ethylene glycol, with its lower molecular weight and higher permeability, would be preferred over glycerol for equine embryos. The purpose of this study was to determine concentrations of glycerol and ethylene glycol within equine blastocoele fluid of Day 9 to 11 equine embryos following 30 min of exposure to a 1.5 M solution. Fourteen grade 1 embryos were recovered from May through September, using standard transcervical lavage techniques. Day of ovulation (Day 0) was determined as first appearance of the corpus luteum by daily transrectal ultrasonography, following insemination and administration of an ovulatory induction agent (Chorulon, 2500 IU; Intervet, Canada). Embryos were graded, measured, and washed in holding media (emP3TM Partnar Animal Health, Ontario, Canada), blocked by age, and randomly assigned to either 1.5 M ethylene glycol in holding solution (EG; n = 7) or 1.5 M glycerol in holding solution (Gly; n = 7) each containing 1% dimethyl sulfone as the internal standard, for 30 min at room temperature. Following CPA exposure, embryos were moved to a sterile, dry dish, all fluid was removed with a micropipette, and the dish dried with fine tissue. The embryonic capsule was punctured, and aspirated blastocoele fluid was analysed for CPA concentration by gas chromatography using a CP-3800 gas chromatograph (Varian Canada Inc., Mississauga, Ontario, Canada). The CPA concentration was determined by comparing the internal standard count to the accumulated CPA in millimoles per liter by gas chromatograph. A two-sample Kolmogorov-Smirnov test for equality of distribution was used because data were not normally distributed. Post-hoc 2-way t-test with unequal variances was used to compare mean effective CPA concentration across treatments, with significance set at P = 0.05. Collected embryos ranged from 1470 μm to 9240 μm in diameter, but there was no difference in embryo size between groups (P = 0.59). Mean concentrations of EG from blastocoele fluid were significantly higher than Gly (71.58 ± 18.9 mmol L–1 v. 50.71 ± 11.2 mmol L–1, respectively; P = 0.016). This represents 4.8 and 3.4% of available CPA, respectively. Mean EG concentrations by embryo age were not different (Day 9: 68.3 ± 23.1 mmol L–1; Day 10: 83.0 ± 15.4 mmol L–1; Day 11: 71.0 ± 21.9 mmol L–1; P = 0.75). Mean Gly concentrations by embryo age were not different (Day 9: 47.0 ± 12.7 mmol L–1; Day 10: 46.0 ± 7.1 mmol L–1; Day 11: 56.3 ± 13.6 mmol L–1; P = 0.35). The low concentrations of EG and Gly found in this preliminary study are in agreement with indirect evidence from other studies. Ethylene glycol reached higher concentrations inside the embryo than glycerol because of lower molecular weight and higher permeability. However, permeation appeared to be minimal for both agents despite exposure to solutions of high CPA molarity.
48 CARBOXYLATED ε-POLY-L-LYSINE (COOH-PLL) IMPROVES DEVELOPMENTAL ABILITY AND REDUCES MITOCHONDRIAL DAMAGE OF VITRIFIED PORCINE EMBRYOS AT THE PRONUCLEAR STAGE
Authors: Kamoshita M. , Fujiwara K. , Matsumura K. , Hyon S.-H. , Ito J. , Kashiwazaki N.
Published: December 2015

Although cryopreservation of pronuclear-stage embryos has increasing importance in the production of genetically modified animals via microinjection, it is well known that cryopreserved porcine embryos show low developmental ability. Cryopreservation causes damage to organelles such as mitochondria that are involved in metabolism and play important roles in embryonic development. To reduce the damage in embryo cryopreservation, we focused on carboxylated ε-poly-l-lysine (COOH-PLL), which has been recently developed as a new cryoprotective agent. Carboxylated ε-poly-l-lysine has antifreeze protein properties, such as inhibition of ice recrystallization (Matsumura and Hyon 2009). In this study, we examined the effect of COOH-PLL on development and mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Porcine follicular oocytes were matured (in vitro) and then fertilized (IVF) in vitro. At 10 h after IVF, the presumptive embryos were centrifuged to visualise the pronuclei. Embryos with 2 or 3 pronuclei (PN embryos) were used for vitrification. The PN embryos were exposed to equilibration solution for 10 min and then exposed to vitrification solution for 1 min before being plunged into LN on Cryotop®. Vitrification solution was phosphate buffered saline supplemented with 30% (vol/vol) ethylene glycol + 0.5 M sucrose + 20% (vol/vol) fetal calf serum + 0 or 20% (wt/vol) COOH-PLL (P0 or P20). Development to the blastocyst stage of the vitrified PN embryos was observed after in vitro culture for 158 h. Nonvitrified embryos were used as a control (fresh). Fresh and vitrified with P0 or P20 PN embryos were stained with MitoTracker® Red CMXRos. The embryos stained with MitoTracker® Red CMXRos were investigated by using a laser-scanning confocal microscope. Mitochondrial fluorescent intensity of embryos was evaluated by measuring the mean numbers of pixels in the fluorescent area (mean/area) using the NIH ImageJ software. The blastocyst rate of embryos vitrified with P20 (19.4%) was significantly (P < 0.05) higher than that of P0 (1.3%), and there were no significant differences in blastocyst rates between P20 and fresh group (28.4%; P > 0.05). The mitochondrial fluorescent intensity of embryos vitrified with P20 (2.8 mean/area) was significantly higher than that of P0 (1.5 mean/area), but the intensity of embryos vitrified with P20 was significantly lower than that of fresh (4.2 mean/area; P < 0.05). Our results suggest that supplementation of COOH-PLL with vitrification solution improved development and also reduced mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Further studies are required to clarify the effect of COOH-PLL on various kinds of damage caused by cryopreservation such as other organelles, including endoplasmic reticulum damage, cytoskeleton damage, or DNA fragmentation of vitrified or warmed embryos in pigs.
57 DEVELOPMENTAL CHARACTERISTICS OF LATER-STAGE PORCINE EMBRYOS PRODUCED IN VIVO OR IN VITRO
Authors: Callesen H. , Holm P.
Published: December 2015

Kinetics and morphological characteristics during pre-implantation embryo development are well established in most mammals. In porcine, such studies are few because of limited use of in vitro culture, but this has changed in recent years due to increasing use of in vitro production and cloning. Therefore, additional characterisation of especially later stage porcine embryos is needed. Here we studied in vitro development of porcine in vivo- and in vitro-derived zygotes collected from weaned, inseminated sows (slaughtered 2 days after first insemination; in vivo group, n = 112) or produced from immature oocytes from sow ovaries (matured and fertilized: Theriogenology 63, 2040–2052; in vitro group, n = 210). Both types were cultured for 7 days in vitro (Theriogenology 63, 2040–2052) in a time-lapse system (Theriogenology 50, 1285–1299) with images recorded every 0.5 h. Individual embryos were followed and characterised for stage and quality from first cleavage. The following was recorded: (i) zygote: inner (i.e. ooplasma) and outer diameter [i.e. including zona pellucida (ZP)], ZP thickness; (ii) compact morula: areas of compacted inner cells and of cellular debris; (iii) blastocyst: partial or total collapse of blastocoelic cavity and diameter at maximal expansion immediately before hatching. At the 1-cell stage, no difference was found between in vivo v. in vitro zygotes in ZP diameter (approximately 150 µm) and thickness (approximately 15.6 µm). In both groups, cleavage rate was around 65%, but more in vivo (85%) than in vitro (28%) zygotes developed beyond the morula stage. Embryos of both types that did not develop to this stage (n = 212) blocked predominantly at the 1st (50%) or 2nd (21%) cell cycle. Cell cycles were generally shorter in in vivo v. in vitro zygotes from compact morula until the hatched blastocyst stage (mean 128 v. 139 h from the 2-cell stage for in vivo v. in vitro; P < 0.05). Compacted in vivo morulae were 25% larger than in vitro, and the debris area was more than twice as large in in vitro. Hatching occurred after approximately two collapses in both in vivo and in vitro but at a larger ZP diameter in vitro. See Table 1 for further details. This study illustrates differences and similarities between morphology and developmental kinetics of in vivo- and in vitro-derived porcine zygotes, but also how various morphological characteristics indicate some of the possible causes of the reduced developmental ability of in vitro embryos. The in vitro period seems to result in more stressful conditions for the embryos, both during early development, but also during the later stages leading to the hatching process. Thus, further optimization of in vitro culture conditions is still needed in porcine.

Table 1.Compact morula (CM) viable cell mass and debris, hatched blastocyst (HB) diameter, and early-hatched blastocyst (EB-XB) and expanded-hatched blastocyst (XB-HB) collapses for in vivo- and in vitro-derived zygotes
59 LIPID DYNAMICS IN THE EARLY EMBRYONIC DEVELOPMENT OF ZEBRAFISH BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY IMAGING AND NANOELECTROSPRAY–MASS SPECTROMETRY
Authors: Jarmusch A. K. , Mahapatra C. T. , Pirro V. , Ferreira C. R. , Sepúlveda M. S.
Published: December 2015

This study introduces a combination of mass spectrometry-based analytical approaches with little or no sample preparation for the study of zebrafish (Danio rerio) lipid metabolism during early development (0 through 96 h post-fertilization, hpf). Desorption electrospray ionization mass spectrometry (DESI-MS) imaging and nanoelectrospray (nESI) MS were used. Embryos (N = 107) were placed onto filter paper squares and analysed by DESI-MS imaging. Embryos were arranged in rows with each one corresponding to a different developmental time, so that each DESI-MS image contained arrays of samples at different hpf. After completion of DESI-MS acquisition, the arrays were cut into strips of paper, isolating each embryo, which was subsequently inserted into a nESI tip and analysed. All experiments were performed using a linear ion trap mass spectrometer. DESI-MS images were acquired in the positive ion mode using acetonitrile doped with 10 ppm AgNO3, which allowed for the detection of cytosolic neutral lipids, such as cholesteryl esters (CE), diacylglicerols (DAG), triacylglycerols (TAG), squalene, and ubiquinone. For the nESI, experiments were run in full mass scan and MS/MS, in both negative and positive ion mode, using a mixture of dimethylformamide and acetonitrile to detect membrane phospholipids (e.g. phosphatidylcholines and phosphatidylglycerols). Principal component analysis was used to explore DESI-MS images and nESI data in an unsupervised fashion. The combination of DESI-MS imaging, providing chemical and spatial location, and nanoESI, providing a broader and more extensive structural information, showed that the lipid content changes dramatically over the first days of development. Accumulation of DAG and TAG, which are usually concentrated in the embryos yolk sac as an important storage for early development, were observed between 0 and 48 hpf. Embryos at the intermediate phase of development (24 hpf) were distributed between those of 0 and 48 hpf, reflecting the dynamics associated with development and might be correlated with TAG consumption and de novo synthesis. After 72 and 96 hpf, samples differed from prior developmental stages by the content of squalene, ubiquinone, and CE 22 : 5 (overexpressed), while the TAG content decreased. Further decrease of TAGs and ubiquinone occurred between 72 and 96 hpf. These observations indicated that zebrafish embryos rely entirely on the yolk sac for the nutrients needed to sustain growth and survival during the first four days of development. Yolk lipids are the likely source of TAGs, as well as cholesterol, a required component of cell membranes and a precursor for bile acids. The abundance of ubiquinone can be tied to zebrafish embryo growth, differentiation and organogenesis, and activation of mitochondria.
63 EQUINE FOLLICLES MODULATE CORTISOL LEVELS AND CAPABILITY OF OOCYTES TO ADAPT TO STRESS SITUATIONS
Authors: Scarlet D. , Ille N. , Gastal G. D. A. , Alves B. G. , Paiva S. O. , Gastal M. O. , Gastal E. L. , Aurich C.
Published: December 2015

Glucocorticoids are mediators of the systemic stress response. Acute or chronic stress characterised by high cortisol concentrations in the periphery impairs reproductive function in a variety of species and therefore may affect fertility. The ovary has been shown to be a target tissue for glucocorticoids in many species, including the mare. This study hypothesised that the equine ovary possesses internal mechanisms to modulate cortisol activity and that supraphysiologic levels of glucocorticoids do not affect oocyte IVM rates. Light horse mares (n = 9) were used in this study. Growing follicles from an induced follicular wave were divided into the following groups: G1: 5–9 mm, G2: 10–14 mm, G3: 15–19 mm, G4: 20–24 mm, and G5: ≥25 mm. Follicular fluid (FF) and compact cumulus‐oocyte complexes (COCs) were obtained by ultrasound-guided transvaginal aspiration. Blood samples were collected at the beginning and the end of every aspiration session. Cortisol (DE1887, Demeditec, Kiel-Wellsee, Germany), progesterone (ADI-901–011, Enzo Life Sciences, Farmingdale, NY, USA), and corticosteroid binding globulin (CBG, MBS047353, MyBioSource, San Diego, CA, USA) concentrations were determined by ELISA. COCs (n = 80) were randomly distributed to either the control group (DMEM-F12+ medium) or the following hydrocortisone treatment groups: 0.1 µg mL–1, 1 µg mL–1, 5 µg mL–1, 10 µg mL–1. Maturation rate was assessed 30 h after incubation. Statistical analysis was performed with the SPSS Statistics 22 software. Data were analysed using one-way ANOVA, Pearson correlation, and chi-squared test. Cortisol (115.4 ± 13.3 ng mL–1) and progesterone (22.1 ± 3.1 ng mL–1) FF concentrations were higher (P < 0.05) in G5 follicles than in all other groups, and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol (118.6 ± 7.8 v. 120.3 ± 12.2 ng mL–1), progesterone (2.4 ± 0.5 v. 2.5 ± 0.4 ng mL–1), and CBG (11.1 ± 5.1 v. 9.9 ± 3.2 µg mL–1) did not differ before and after follicle aspiration. However, plasma CBG and progesterone were negatively correlated (r = –0.56; P < 0.01). Maturation rates did not differ among groups, regardless of the hydrocortisone concentration added to the culture medium. Our results demonstrated higher cortisol concentrations in preovulatory follicles in vivo, suggesting its importance for oocyte maturation. The greater unbound cortisol available in the FF of preovulatory follicles can be indicative of the displacement of cortisol from CBG in favour of progesterone. Furthermore, equine oocytes were capable of surviving cortisol concentrations 100 times higher than those physiologically present in preovulatory follicles. This finding suggests the ability of equine oocytes to modulate cortisol levels and adapt to stress situations.
7 KISSPEPTIN SLOWLY ADMINISTERED DURING PROESTRUS IMPROVES FOLLICLE GROWTH AND OVULATION IN ANESTROUS COWS
Authors: Macedo G. G. , Sala R. V. , Martins L. F. , Baruselli P. S.
Published: December 2015

This study evaluated whether kisspeptin (Kp) administered slowly from the moment of prostaglandin F2α (PGF) treatment for fixed-time AI protocol of anestrous cows provides suitable dominant follicle (DF) growth similarly to eCG. Postpartum, anestrous, primiparous Nelore cows (n = 50; <8-mm follicles with no corpus luteum) and lean (body condition score 2.7 ± 0.1; 0–5 score scale) were enrolled. On Day 0, females received an intravaginal device with 1.0 g of progesterone (Sincrogest®, Ourofino, Brazil) and 2 mg of oestradiol benzoate IM (Sincrodiol®, Ourofino, Brazil). On Day 8, the device was removed, and cows were administered 500 μg of sodic cloprostenol IM (Sincrocio®, Ourofino, Brazil) and 1 mg of oestradiol cypionate IM (E.C.P.®, Zoetis, Brazil). Females were blocked by dominant follicle diameter and allotted in following groups: (1) control (CTRL), 1.5 mL of saline IM; (2) eCG, 300 IU IM of eCG (Novormon®, Intervet, Brazil); and (3) Kp SC with an osmotic minipump (9.65 μg kg–1 of Kp diluted in saline) over 48 h. Dominant follicle growth and ovulation evaluation were performed by ultrasound at 0, 24, 48, 60, 72, 84, and 96 h after PGF treatment. Dominant follicle growth rate was calculated by difference on DF from 0 to 48 h. Ovulation dispersion was analysed by proc GLIMMIX of SAS (SAS Institute Inc., Cary, NC, USA). Follicle variables, ovulation rate, and interval from PGF to ovulation were analysed by orthogonal contrast. Differences were considered when P < 0.05, and tendencies were considered when P < 0.1. There were no difference between CTRL and growth promoters (eCG and Kp) or between growth promoters on DF diameter at 0 h, larger follicle registered from 0 h to ovulation, ovulatory DF diameter, ovulation rate, and interval between PGF treatment and ovulation (Table 1). Dominant follicle diameter at 48 h was larger for growth promoters than for CTRL. Treatment with Kp proved as efficient as eCG in the DF growth rate, with tendency (P = 0.1) to be greater than CTRL. Data from our laboratory (Sales et al. 2011 Anim. Rep. Sci. 124, 12–18.) with same model and animal profile (n = 150 per group) presented positive effect of eCG treatment for growth and size of DF. Cows treated with Kp ovulated in only 3 specific times (60, 72, and 84 h), unlike eCG (48, 60, 72, and 84 h) and CTRL (24, 48, 60, 72, 84, and 96 h; P > 0.05). Also, it is noted that Kp treatment dose and releasing does not cause negative effect on follicle growth and ovulation (down-regulation on LH releasing) or alteration on interval of PGF treatment to ovulation. Thus, synchronized anestrous cows treated with Kp delivered slowly during proestrus have similar results to eCG on follicle growth and ovulation.

Table 1.Ovarian variables of anestrous Nelore cows treated with saline, eCG (300 IU), or kisspeptin (Kp; 9.65 μg kg–1). Data were analysed by orthogonal contrast, where C1 = growth promoter (eCG and Kp) v. control; C2 = eCG v. Kp