bluuebunny/crossref_metadata_2025 huggingface dataset:
157 EFFECT OF DENSITY GRADIENT SELECTION ON EMBRYO RECOVERY RATES OF FRESH AND COOLED STALLION SEMEN
Authors: Papa F. , Carmo M. , Papa P. , Dell'Aqua J. , Alvarenga M.
Published: December 2012

The aim of this study was to improve the spermatic kinetic parameters from stallions with poor quality of fresh and chilled semen by the use of density gradient Equipure™ (Nidacon, Mölndal, Sweden). Semen from 5 Quarter Horse stallions aged 8 and 16 years with history of low embryo recovery rates were used. The kinetics sperm evaluation was performed by computerized semen analysis (CASA) and plasma membrane integrity with fluorescent probes. The average motility parameters for fresh semen before selection were total motility (MT) 60%, progressive motility (PM) 30%, and plasma membrane integrity (IMP) 60% and for cooled semen (24 h at 5°C) were: MT 50%, MP 18%, and 50% IMP. For the group of fresh and cooled semen with no density gradient selection (NS), mares were inseminated with 1 billion viable sperm diluted in skim milk extender in the uterine body, 24 h after ovulation induction with 1 mg of deslorelin. For EquipureTM selection group (SE) semen was concentrated through Spermfilter membrane™ and resuspended in 5 mL of BotuSemenTM (BotupharmaTM, Brazil). In a 15 mL conic tube 5 mL of EquipureTM was added and another part containing 5 mL of the resuspended sperm was slowly added over the EquipureTM column. The 15 mL conic tube was centrifuged at 400g for 20 min. After centrifugation, the sperm pellet was carefully aspirated and to the pellet was resuspended in 4 mL of BotucrioTM. The sperm recovery rate with EquipureTM was 40%. Deep uterine AI was performed 24 h after ovulation induction with 1 mg of deslorelin. Data were analyzed by ANOVA followed by Tukey’s test (P < 0.05). The analysis of semen after EquipureTM selection resulted in average: 75, 35, 65, and 40%, respectively, for MT, MP, and IMP index and sperm recovery. The embryo recovery rate from the 5 stallions showed the following results: Stallion 1 (fresh semen): 12 mares (NS)/4 embryos (33%) and for group SE 22 mares/16 embryos, (72%; P ≤ 0.05). Stallion 2 (fresh semen): 9 mares (NS)/4 embryos, (44%) and for group SE, 12 mares/8 embryos (66%; P > 0.1). Stallion 3 (fresh semen): 4 mares (NS)/1 embryo (25%) and 7 mares for SE/4 embryos (57%; P > 0.1). Stallion 4 (chilled semen): 4 mares (NS)/0 embryo 0% and for SE group 8 mares/6 embryos 75%; P ≤ 0.001). Stallion 5 (chilled semen): 10 mares (NS)/2 embryos (20%) and for SE 6 mares/3 embryos (50%; P > 0.1). For the overall results, 39 inseminations were performed on the no selected group and 11 embryos recovered (28%) for the selected group 55 inseminations and 37 embryos recovered (67%; P < 0.01). The results clearly showed that selection by sperm density gradient EquipureTM was a very effective technique that allowed an improvement on semen quality and fertility.
The authors acknowledge support from FAPESP and Botupharma.
179 CORPUS LUTEUM DEVELOPMENT AFTER FOLLICLE ASPIRATION IS RELATED TO CHARACTERISTICS OF THE ASPIRATED FOLLICLE IN DAIRY COWS
Authors: Vernunft A. , Weitzel J. M. , Viergutz T.
Published: December 2012

The aim of this study was to investigate whether morphology or function of a corpus luteum (CL), which developed after an aspiration of a preovulatory follicle, is related to follicular characteristics such as size or steroid content. If so, CL morphology or function could be used as a retrospective quality parameter for the aspirated follicle or the follicular characteristics as a prospective parameter for the quality of the developing CL. Therefore, 44 aspiration sessions were performed using 18 cows between 26 and 121 days after parturition during the first lactation. Heat was induced in mid-dioestrus with Cloprostenol. A GnRH analogon (Depherelin) were administrated 54 h later. The dominant follicle was aspirated 21 h after administration of the GnRH analogue. The diameter of the dominant follicle at aspiration and the cross-section area of the resulting luteal tissue 14 days later were measured by ultrasound. Concentrations of progesterone (P4) and 17-β-oestradiol (E2) in blood and follicular fluid (FF) were determined by H3-RIA. A CL development occurred in 82% of aspiration sessions after aspirating the dominant follicles. The interval of time between parturition and follicle aspiration did not affect variables investigated. As expected, cross-section area of the luteal tissue was positively correlated with plasma P4 concentration on Day 14 after follicle aspiration (r = 0.54; P < 0.01). The diameter of the aspirated follicle was positively correlated with the plasma P4 concentration on Day 14 after follicle aspiration (r = 0.52; P = 0.02), but the diameter was not correlated with the cross-section. Comparing the FF of follicles that originated a CL after aspiration with follicles that did not, both types had comparable P4 concentrations (578 ± 79 ng mL–1 v. 398 ± 84 ng mL–1; P = 0.2), but the former type presented higher E2 concentrations (206 ± 23 ng mL–1 v. 64 ± 29 ng mL–1, P < 0.01) compared with the latter. The P4 concentrations in FF of follicles that originated a CL after aspiration were positively related to cross-section area of the CL (r = 0.48; P = 0.04), but neither P4 nor E2 concentrations in the FF of preovulatory follicles could be used as a predictor for the plasma P4 concentration at day 14. In conclusion, CL development after follicle aspiration can be used as a retrospective quality parameter of aspirated dominant follicles. Preovulatory follicle diameter as well as cross-section area of the luteal tissue could be used prospectively to identify cows with high plasma P4 levels, and this may help to identify suitable recipients for embryo transfer.
This study was supported by the German Research Foundation (DFG WE 2458/7-2).
183 EFFECTS OF LONG-TERM NUTRITIONAL RESTRICTION ON PREOVULATORY LUTEINIZING HORMONE SURGE, LEPTIN, AND LUTEAL PHASE IN LLAMAS (LAMA GLAMA)
Authors: Norambuena M. C. , Silva M. , Urra F. , Ulloa-Leal C. , Letelier C. , Fernández A. , Adams G. P. , Ratto M. H.
Published: December 2012

The aim of the study was to determine the effect of long-term energy restriction on preovulatory LH surge and luteal phase in llamas. Mature nonlactating, nonpregnant, female llamas (n = 16) were assigned randomly to the following groups: (1) llamas received Ballica sp. hay ad libitum and 300 g d–1 of commercial concentrate (control group, n = 8, BCS = 3.9) or (2) llamas were fed the same feed but the amount was progressively reduced from 70% to 40% of their maintenance energy requirement (MER) for a period of 87 days, until the final BCS was 2.5 (restricted group, n = 8). After 2 months and 21 days of 40% MER diet, ovarian follicular wave emergence was synchronized by transvaginal ultrasound-guided ablation of follicles ≥5 mm in diameter using a 17-ga needle attached to a 5-MHz convex-array transducer. Ten days after follicle ablation (Day 0), llamas were given an i.m. dose of 50 µg of gonadorelin acetate to induce ovulation. Frequent blood samples were taken every 30 min for 6 h and every 15 min for 1 h for plasma LH and leptin concentration measurements, respectively, immediately after treatment. Llamas were examined daily by transrectal ultrasonography for 20 days to record serial changes in the CL diameter. In addition, blood samples were taken every 2 days from Day 0 to 16 to determine plasma concentrations of leptin and progesterone. Discrete dependent variables were compared between groups by Student t-tests and continuous dependent variables were analysed by analyses of variance with repeated-measures. There was an effect of time on plasma LH concentration (P ≤ 0.001), but not an effect of group (P = 0.1) or interaction (P = 0.6) after gonadorelin acetate treatment. Plasma leptin concentration was lower in llamas from the restricted than that of the control group after gonadorelin treatment [effect of time, group (P ≤ 0.01), and interaction (P ≤ 0.01)]. The maximum diameter of the CL and plasma progesterone concentration were lower in the restricted than in the control group, and both variables were affected by group (P ≤ 0.01, P ≤ 0.02), and day (P ≤ 0.001, P ≤ 0.001, respectively). Plasma leptin concentration was lower in the restricted than that of the control group during the entire experiment. In conclusion, long-term nutritional restriction affected negatively CL diameter, progesterone secretion, and leptin plasma concentration in llamas. The differential response of leptin to gonadorelin acetate treatment could reflect a subfertlity-nutritional induced problem related with steroidogenesis, oocyte quality, ovulation, and early embryo development processes.
Research supported by Postdoctoral Fondecyt N° 3110095.
191 RISK ASSESSMENT OF BISPHENOL A, AN ENDOCRINE DISRUPTOR, VIA PROLIFERATIVE EFFECT ON THE GROWTH OF ESTROGEN-DEPENDENT OVARIAN CANCER IN CELLULAR AND ANIMAL MODELS
Authors: Hwang K.-A. , Choi K.-C.
Published: December 2012

One of estrogens in the body, 17β-oestradiol (E2), is a pleiotropic hormone that regulates the growth and differentiation of many tissues and also acts as a mitogen that promotes the development and proliferation of hormone-responsive cancers such as breast and ovarian carcinomas. Xenoestrogens are chemical compounds that imitate oestrogen in living organisms and are classified as a type of endocrine-disrupting chemical (EDC). Bisphenol A (BPA) is a widely used industrial compound, and also known as an EDC and especially a xenoestrogen. In this study, we examined the effect of E2 or BPA on the cell growth of BG-1 ovarian cancer cells in vivo and in vitro. In the cell proliferation assay in vitro, E2 or BPA increased the growth of the BG-1 ovarian cancer cells expressing oestrogen receptors (ER). Their proliferation activity was reversed by the treatment of ICI 182 780, a well-known antagonist of ER, which demonstrates that the cell proliferation by E2 or BPA is mediated by ER and BPA certainly acts as a xenoestrogen in the BG-1 ovarian cancer cells. Clearly, E2 and BPA increased the expression of cyclin D1, a factor responsible for the G1/S cell cycle transition. These reagents also decreased the expression of p21, a potent cyclin-dependent kinase (CDK) inhibitor that arrests the cell cycle in the G1 phase. As a result, they promoted the proliferation of BG-1 cells via upregulation of the cell cycle progression. In mice xenograft models transplanted with BG-1 ovarian cancer cells, E2 or BPA administration significantly induced the tumour proliferation compared with vehicle (corn oil) treatment for 10 weeks, which was identified by the measurement of tumour volume and histological analysis on tumour tissues such as hematoxylin and eosin (H&E) staining and BrdU incorporation assay. Taken together, as an EDC having a xenoestrogenic activity, BPA was demonstrated to have a risk of tumour proliferation in oestrogen-dependent cancers such as ovarian cancer.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) of government of Korea (no. 2011-0015385).
2 TRANSCRIPTOME ANALYSIS OF GRANULOSA CELLS FROM GROWING DOMINANT FOLLICLE REVEALS AGE-ASSOCIATED CHANGES AT THE TIME OF FOLLICLE SELECTION IN AGED BEEF CATTLE
Authors: Khan M. I. R. , Dias F. C. F. , Sirard M. A. , Adams G. P. , Singh J.
Published: December 2012

Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA; Ingenuity Systems Inc., Redwood City, CA) analysis of these transcripts revealed that granulosa cells of aged cows exhibit (1) reduced capability to regulate gonadotropins [↓follistatin (FST), ↓inhibin beta A (INHBA), ↓inhibin beta B (INHBB)] and reduced responsiveness to gonadotropin-induced changes in cytoskeleton [↑tropomyosin 2 (TPM2), ↑actin gamma 2 (ACTG2), ↓tubulin beta] and extracellular matrix [↓tumor necrosis factor alpha-induced protein 6 (TNFAIP6), ↓versican (VCAN)], (2) inefficiency in processing lipids [↓low-density lipoprotein receptor (LDLR), ↓stearoyl-coenzyme A desaturase (SCD), ↑cluster of differentiation 36 (CD36), ↓sterol-C4-methyl oxidase-like (SC4MOL)] and synthesizing steroids [↓cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), ↓cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1)], (3) decreased proliferation [↓proliferating cell nuclear antigen (PCNA)] and control of cell cycle check points [↓checkpoint kinase 1 (CHEK1), ↓centromere-associated protein E (CENPE)] and have poor intercellular communication [↓gap junction protein alpha 1 (GJA1)], and (4) higher expression of oxidative stress responsive genes [↑vanin-1 (VNN1), ↑vanin-2 (VNN2), and ↑glutathione peroxidase 3 (GPX3)]. A total of 6 transcripts: CYP19A1 (aromatase; P ≤ 0.1), VNN1 (P ≤ 0.05), INHBA (P ≥ 0.1), PCNA (P ≤ 0.001), TPM2 (P ≤ 0.1), and GJA1 (P ≤ 0.05) were selected to validate the microarray results via quantitative real-time PCR. In conclusion, granulosa cells of growing dominant follicles exhibit age-related changes in the transcriptome at the time of selection relative to young cows; changes that may explain follicle-associated loss of oocyte competence in aged cows.
Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada).
205 RNA-SEq PROFILING OF BOVINE PRE-IMPLANTATION EMBRYOS
Authors: Krebs S. , Graf A. , Valeri Z. , Blum H. , Wolf E.
Published: December 2012

In order to provide a comprehensive view of the transcriptome changes during the earliest stages of bovine development, we sequenced the total RNA content of bovine oocytes, 4-cell, 8-cell, and 16-cell embryos and the inner cell mass and trophoblast envelope of expanded blastocysts on the Illumina Genome Analyzer IIx. For each experiment pools of in vitro matured oocytes from the German Simmental cows were fertilized by sperm of a single bull, and 10 oocytes or embryos per developmental stage were collected to generate total RNA pools used for sequencing. Synthesis of cDNA was initiated directly in the cell lysate in order to avoid any losses during RNA preparation and was random primed in order to capture all RNA species. Amplified cDNA and unstranded sequencing libraries were prepared using kits from Nugen (Ovation RNA-Seq, Nugen, San Carlos, CA, USA). Biological replicates were generated by inseminating the oocytes with sperm from the distant breeds Jersey (n = 3) and Brahman (n = 3). This cross-breeding design allowed tracking of single sequencing reads back to the maternal or paternal genome, where breed-specific SNP are present in the expressed transcripts. The analysis of this dataset resulted in monitoring of zygotic genome activation and parent-specific expression for single transcripts, a catalogue of splicing isoforms, novel transcripts, and non-coding RNAs and differentially expressed genes between the single developmental stages. Using the program DESEqn, 2784 genes showed differential expression between any of the stages at a false discovery rate of 1%. Specifically, we found 200 genes differentially expressed between immature and matured oocytes, 209 genes between matured oocytes and 4-cell embryos, 580 genes between the 4-cell and 8-cell stage, 567 genes between the 8-cell and 16-cell stage, 987 genes between the 16-cell stage and the inner cell mass, and 1569 genes between the 16-cell and the trophoblast. Functional analysis revealed stage-specific functions of the differentially expressed genes. In summary, by fully exploiting the single-nucleotide resolution of the RNA-Seq method, this dataset provides an invaluable resource for the study of zygotic genome activation, imprinting, transcript annotation, and gene expression in the earliest developmental stages of bovine embryos.
211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER
Authors: Gad A. , Besenfelder U. , Havlicek V. , Hölker M. , Cinar M. U. , Rings F. , Dufort I. , Sirard M. A. , Schellander K. , Tesfaye D.
Published: December 2012

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.
229 RAPID, UNTARGETED LIPID DETERMINATION IN INDIVIDUAL BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS BY HIGH-RESOLUTION DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY
Authors: González-Serrano A. F. , Ferreira C. R. , Pirro V. , Eberlin L. S. , Heinzmann J. , Lucas-Hahn A. , Niemann H. , Cooks R. G.
Published: December 2012

Lipid structural analysis in individual pre-implantation mammalian embryos is hampered by the small amount of biological material, such that most studies use staining methods or gas chromatography analysis generate information only on the fatty acyl residues. Recent developments in high-resolution desorption electrospray ionization mass spectrometry (DESI-MS) allow the analysis of free fatty acids (FA) and glycerophospholipids (PL) in individual bovine embryos. Here, we report on the use of DESI-MS for the sensitive analysis of triacylglycerol (TAG) species, profiles of FA and PL in individual bovine oocytes and embryos. Bovine oocytes (n = 40) and blastocysts (n = 42) were frozen in a minimal volume of PBS (2 to 5 µL). Samples were directly deposited on glass slides after thawing. After drying, a volume of 500 µL of methanol : water (1 : 1, vol/vol) was carefully deposited on the surface of the glass slide and removed by orienting the glass slide vertically to eliminate PBS salts. An Orbitrap mass spectrometer was used for the experiments. Parameters for the positive ion mode were as follows: acetonitrile (ACN) supplemented with 3 µL mL–1 of AgNO3 at a 5 µL min–1 flow rate, injection time of 1000 ms, and a mass-to-charge range of m/z 400 to 1500. For the negative ion mode, the solvent combination used was acetonitrile + dimethylformamide (1 : 1, vol/vol) at a 1.0 µL min–1 flow rate, a maximum injection time of 1000 ms, and a mass-to-charge range of m/z 150 to 1000. Positive ion mode data for the detection of TAG species were acquired first, followed by acquisition of FA and PL in the negative ion mode. Detection of TAG by DESI, which is extremely useful for bovine embryo cryopreservation and metabolism research, has been performed by adding AgNO3 in the DESI spray to obtain silver adducts, which are easily recognised by the characteristic 1 : 1 abundance ratio of the 107 : 109 Ag isotopes. The most abundant fatty acyl residues present in TAG species were palmitic (P), linoleic (L), oleic (O), and stearic (S) acids, such as TAG of m/z 937, PPL (50 : 2); m/z 965, POO (52 : 1); m/z 967, POS (52 : 2); m/z 989, OOL/LLS (54 : 4); and m/z 991, OOO, SOL (54 : 3). Free FA and PL profiles collected from the same samples in the negative ion mode were similar to those in our recent report (2012 J. Mass Spectrom. 47, 29–33). Lipid attribution has been performed based on high-resolution mass analysis. Multivariate statistics from this data set will allow visualisation of differences observed in the lipid profiles among samples. In conclusion, we report the use of DESI-MS for the sensitive analysis of TAG in individual bovine oocytes and embryos and the creation of profiles of FA, PL, and TAG species in the same sample by DESI-MS.
231 EFFECT OF DIFFERENT EQUINE CHORIONIC GONADOTROPIN CONCENTRATIONS ON IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Decanine C. , Pioltine E. M. , Emanuelli I. P. , Puelker R. Z. , Nogueira M. F. G.
Published: December 2012

In vitro maturation (IVM) is one of the most challenging steps in the in vitro production of bovine embryos. The IVM medium must provide the necessary conditions for the occurrence of nuclear and cytoplasmic maturation, close to the physiological conditions. The pituitary gonadotropins are essential components for generating competent oocytes; however, whether these hormones (pituitary, placental, or both) are essential and which concentrations should be used are still controversial. Our work aimed to compare the effect of different concentrations of the placental gonadotropin (eCG) in the IVM medium on the in vitro-produced bovine blastocysts. Cumulus–oocyte complexes (n = 1341, grades I and II), obtained from ovaries from an abattoir were selected and distributed into six groups: (1) eCG (4 IU mL–1; n = 192); (2) eCG (1.5 IU mL–1; n = 204); (3) eCG (1.3 IU mL–1; n = 203); (4) eCG (0.15 IU mL–1; n = 202); (5) eCG (0.015 IU mL–1; n = 199); (6) control: FSH (0.1 mg mL–1), LH (50 µg mL–1), and 10% of fetal calf serum (FCS; n = 341). Medium from groups 1 to 5 also contained LH (50 µg mL–1) and BSA (6 mg mL–1). The cumulus–oocyte complexes were matured in TCM-199 for 24 h and were IVF (Day 0) in TALP-IVF for 22 to 24 h. Viable spermatozoa were selected by Percoll gradient, and they were evaluated (motility and spermatozoa concentration) to provide the insemination concentration (106 spermatozoa mL–1). Presumptive zygotes were cultured in SOF medium supplemented with FCS (2.5%) and BSA (5 mg mL–1) in an incubator (38.3°C, 5% CO2, and maximum humidity). Embryo development was evaluated in terms of cleavage (Day 3), blastocyst (Day 7), and hatching rates (Day 10). Mean rates were analysed by chi-squared test and ANOVA, and significance was considered when P < 0.05. The results obtained from the different groups are shown in Table 1. Cleavage, blastocyst, and hatching rates were not different among groups. We conclude that, under our experimental conditions, even minimal concentrations of eCG (0.015 IU) may be a viable and effective alternative in the replacement of FSH for the IVM of bovine oocytes.

Table 1.Cleavage, blastocyst, and hatching rates of the experimental groups (1, 2, 3, 4, and 5) and control group1

Fellowships and support by CAPES and FAPESP.
235 PROGESTERONE AFFECTS THE MESSENGER RNA EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Knauer K. , Stinshoff H. , Wilkening S. , Wrenzycki C.
Published: December 2012

It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus–oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100 ng of P4 on Days 4 or 5 of IVC (IVF = Day 0). Alcohol (ETOH) was used as the solvent, so 8 µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at –80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P ≤ 0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20 ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10 ng, 100 ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20 ng of P4 resulted in a significantly higher level of transcripts than when 10 ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected.

Table 1.Cleavage and development rates (± SEM) of embryos supplemented with 10, 20, or 100 ng on Day 4 or 5 of in vitro culture (P ≥ 0.05)

The financial support of the FBF e.V. is acknowledged.
24 EVALUATION OF THE PREGNANCY RATE OF FIXED-TIME ARTIFICIALLY INSEMINATED BOS INDICUS COWS USING HETEROSPERMIC AND MONOSPERMIC SEMEN
Authors: Detomini J. P. , Tirloni R. R. , Araujo C. V. , Martins L. R.
Published: December 2012

The aim of this study was to establish a comparison between heterospermic and monospermic semen on the pregnancy rate of fixed-time AI Bos indicus cows. The experiment was conducted in the state of Mato Grosso, Brazil, during December 2010 and February 2011. Multiparous (n = 173) and primiparous (n = 204) Nelore cows, 45 ± 15 days postpartum, were evaluated on body condition score (BCS; score 1 to 9), and those that presented BCS between 5 and 8 were selected. Two protocols of estrous cycle synchronization were performed (PeCG and Pshang). Synchronization of the estrous cycle was achieved with the aid of the following protocol: on Day 0, all animals were treated with a progesterone vaginal implant with 1.9 g of progesterone combined with an IM injection of 2 mg of estradiol benzoate. On Day 7, an IM injection of 12.5 mg of dinoprost was administered. On Day 9, implants were withdrawn and the animals received an IM injection of 0.5 mg of estradiol cypionate. On Day 9, part of the animals (n = 222) received an IM injection of 300 IU mg of eCG (PeCG) and the other part (n = 155) had their calves removed for 48 hours (Pshang). All animals were inseminated 46 to 52 h after the end of the protocol. Cows were allocated randomly in 4 groups according to the donor male sample in the semen straw and groups were designated as MA (monospermic straw from bull A; n = 110), MB (monospermic straw from bull B; n = 102), MC (monospermic straw from bull C; n = 108), and HT (heterospermic sample from bulls A, B, and C; n = 57). Commercially available semen straws from Aberdeen Angus donors were used (ABS Pecplan, Brazil). Pregnancy diagnosis was performed 35 days after AI using real-time ultrasonography. Results are summarized in Table 1. There was no statistical difference between MA, MB, MC, and HT in any group according to the chi-square test (P > 0.05). However, there was a remarkable difference in pregnancy rates among the 3 bulls tested considering both protocols, probably due to the variation in the ovulation moment.

Table 1.Pregnancy rates of Nelore cows submitted to FTAI protocol inseminated with monospermic (bull A, B, or C) or heterospermic (HT) semen
25 TIMED ARTIFICIAL INSEMINATION ENABLES HIGH FERTILIZATION RATES IN NORMAL CYCLING AND IN SUPEROVULATED CATTLE WITH REDUCED DOSAGES OF UNSORTED SPERMATOZOA AND WITH SEXED SPERMATOZOA
Authors: Becker F. , Kanitz W. , Nurnberg G. , Rath D.
Published: December 2012

Different factors determine the effectiveness of the use of sires in AI. Most important factors are the number of inseminated spermatozoa, the quality of spermatozoa, and the time of insemination. Especially in superovulated animals, the insemination scheme plays in important role to cover the whole ovulation period. The influence of 3 different dosages of spermatozoa (15 × 106, 5 × 106, and 1 × 106) on fertilization rate was examined in experiment A. In experiment B, one dosage of female and male spermatozoa of 3 different bulls was used for timed AI in 31 heifers. Timed AI in normal-cycling cattle [13 h after gonadotropin-releasing hormone (GnRH) application] with detected corpus luteum (Days 8 to 13 of cycle) was carried out after induction of luteolysis and induction of ovulation [GnRH application 60 h after prostaglandin F2α (PGF2) application]. Embryos and oocytes were flushed from the oviduct of 116 hemicastrated or slaughtered heifers on Day 4 after insemination. The ovulation rate in heifers was 95.4%. Eighty percent of the oocytes or embryos were recovered. The influence of the factors sire, ejaculate, and dosage were tested by GLM analyses of SAS® (SAS Institute Inc., Cary, NC, USA). There was no significant difference in the fertilization rate (93.3, 96.2, and 78.8%) and in the proportion of normally developed embryos (84.6, 80.7, and 75.8%) between groups. Significant differences were found in the mean number of accessory sperms/embryo and in the proportion of embryos with >10 accessory sperms/embryo or without accessory sperms; however, the proportion of intact embryos was similar. Using sexed semen in experiment B, similar results were obtained after flushing of the oviducts on Day 4 after insemination of hemicastrated or slaughtered animals. In total, an ovulation rate of 91.7%, a recovery rate of 70%, and a fertilization rate of 86.8% were obtained. There were no differences between female- and male-sorted spermatozoa and the control group. In experiment C, altogether 13 heifers were treated 8 times with FSH for 4 days starting between Day 8 to 12 of estrous cycle. Prostaglandin F2α was given 48 and 60 h after the first FSH injection. Insemination with sexed semen (n = 5 heifers) and with unsorted semen (n = 8; 15 × 106 and 1 × 106) was done at 55 and 71 h after induction of luteolysis. Flushing of the uterus was performed on Day 7. Using the time-oriented insemination after superovulation of animals, fertilization rates varied between 65 and 85%. There was no difference between groups regarding the number of transferable embryos (5.5, 4.9, and 4.8). The results demonstrate that the application of an approved insemination schedule may accomplish high fertilization rates after insemination with sexed or reduced dosages of spermatozoa in normal-cycling as well as superovulated cattle.
250 ALL REGIONS OF THE MOUSE EPIDIDYMIS ARE ABLE TO PHAGOCYTIZE IMMATURE SPERMATOGENIC CELLS
Authors: Ramos-Ibeas P. , Pericuesta E. , Fernandez-Gonzalez R. , Ramirez M. A. , Gutierrez-Adan A.
Published: December 2012

Successful mammalian fertilization requires gametes with an intact structure and functionality. Although it is well known that epididymal functions are sperm maturation, sustenance, transport, and storage, there is controversial information about its role in sperm quality control, and it has been suggested that some regions of the rat epididymis are able to phagocytize germ cells. Our objective was to analyse whether different segments of the mouse epididymal epithelium act as a selection barrier for abnormal spermatogenic cells by removing immature cells from the lumen by phagocytosis. To detect the presence of immature germ cells along the epididymis, transgenic mice expressing enhanced green fluorescent protein under a Deleted in Azoospermia-Like (mDazl) promoter were generated. The transgenic animals express specifically enhanced green fluorescent protein in spermatogonias, spermatocytes, and spermatids; thus, immature spermatogenic cells can be easily identified by fluorescence microscopy. Colchicine, a microtubule disruptor that leads to severe alterations in the architecture of the seminiferous tubules, was administered in the rete testis to induce the release of immature germ cells into the epididymis. Mice were killed daily, from Day 1 to 8 post-administration, and epididymides were collected and observed under a fluorescence stereoscope to determine the transit of immature germ cells along the epididymis. Epididymides from control mice without colchicine administration were also collected. Fluorescent immature germ cells were present in the caput epididymis 24 h after colchicine administration, and they progressed through the corpus and cauda, leaving the epididymis 7 days after colchicine administration. After fluorescence observation, epididymides were fixed, sectioned, and stained with hematoxylin solution. Immature germ cells and phagosomes were not observed in control epididymides. By contrast, the presence of phagosomes in the principal cells of the epididymal epithelium containing immature germ cells in different degrees of degradation was observed by light microscopy in mice injected with colchicine. Phagocytosis was observed along the epididymis following the main wave of fluorescent immature cells. Thus, when immature cells had reached the corpus epididymis, phagocytosis was detected in several segments of the caput epididymis. Later, once the immature cells had arrived to the cauda epididymis or had abandoned the epididymis, phagocytosis was observed in the corpus and cauda epididymis. The presence of phagosomes was observed in all epididymal tubules within a phagocytosis area. In conclusion, we demonstrated that the epididymal epithelium is engaged in sperm quality control by clearing immature germ cells after a massive shedding into the epididymal lumen, and that this phenomenon is not restricted to a specific segment of the epididymis.
262 MEIOTIC SPINDLE CONFORMATION ASSESSMENT BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES
Authors: Caamaño J. N. , Cuadrado F. , Díez C. , Muñoz M. , Martín D. , Carrocera S. , Gómez E.
Published: December 2012

Polarized light microscopy (PLM) allows detection of microtubule-polymerized protein in in vitro-matured sheep and goat oocytes (Caamaño et al. 2011 Reprod. Fertil. Dev. 23, 226–227). Spindle birefringence, measured as the mean retardance value, has been proposed as a marker of meiotic spindle conformation in humans and mice. Because the conformation of the meiotic spindle cannot be readily assessed by PLM, in this study we aimed to measure the mean retardance value of normal and abnormal meiotic spindles from in vitro-matured prepubertal sheep and goat oocytes. Oocytes were matured in vitro for 27 h and were then individually assessed by PLM (Oosight System, Research Instruments Ltd., Falmouth, Cornwall, UK), and the mean retardance value was analysed in meiotic spindles using specific software (Oosight). Meiotic spindle conformation was determined in individual oocytes by immunostaining and chromatin detection, as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191–201). A barrel-shaped spindle was considered a normal spindle configuration. Only oocytes with meiotic spindles identified by both PLM and immunostaining were used in this study. The experiment was replicated four times. Data were analysed by the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). A normal barrel-shaped spindle was observed in 80.8% of the sheep oocytes (n = 47) and in 83.9% of the goat oocytes (n = 62). In sheep, the mean retardance value in oocytes with a normal meiotic spindle conformation did not differ from that in oocytes with abnormal spindles (4.42 ± 0.26 nm v. 3.92 ± 0.54 nm). Similar results were obtained with goat oocytes with normal (2.94 ± 0.20 nm) v. abnormal spindle conformation (2.77 ± 0.08 nm). These results indicate that the mean retardance value does not distinguish between oocytes with normal and abnormal meiotic spindle conformation, as assessed by subsequent immunostaining.
Grant support from INIA-RZ2007-00013-00-00. M. Muñoz was sponsored by RYC08-03454.
265 HYALURONAN SYNTHESIS ABILITY OF PORCINE CUMULUS - OOCYTE COMPLEXES DERIVED FROM SMALL FOLLICLES
Authors: Nakakoji M. , Funahashi H.
Published: December 2012

The degree of cumulus expansion, an important step in oocyte maturation, of porcine cumulus–oocyte complexes (COC) derived from small follicles (SF: 1 to 2 mm in diameter) is known to be lower than those derived from middle follicles (MF: 3 to 6 mm in diameter). The objective of this study was to compare the abilities of hyaluronan (HA) synthesis of COC from SF and MF. Furthermore, the effect of oestradiol during pre-incubation of COC on proliferation of the cumulus cells was examined. Cumulus–oocyte complexes from SF and MF of porcine ovaries were cultured for in vitro maturation [IVM, in modified porcine oocyte medium (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) supplemented with 50 µM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dbcAMP for 20 h and then in the fresh medium without those supplements for another 24 h]. Hyaluronan production was quantified at 20 h after the start of IVM with a commercial HA-ELISA kit (20 COC/tube × 4 times). The number of cumulus cells was assessed 0 and 20 h after the start of IVM (50 COC × 4 times). Furthermore, proliferation of cumulus cells was examined after pre-culture of COC (n = 40 COC × 5 times) in modified porcine oocyte medium with various concentrations of oestradiol (0, 0.1, 1, and 10 ng mL–1) for 6 h. Statistical analyses of results from 4 to 5 replicated trials were performed by ANOVA with a Bonferroni-Dunn post-hoc test (significance, P < 0.05). The degree of cumulus expansion of COC from MF (n = 152) was higher than that of COC from SF (n = 156). The incidence of metaphase-II oocytes was significantly lower in COC from SF (n = 133; 48.9%) than in COC from MF (n = 148; 74.7%). The HA content of COC was higher in those from MF (20.8 µg/COC) than in those from SF (10.8 µg/COC), whereas the content per cumulus cell was not different because the numbers of cumulus cells at 0 and 20 h were also higher in COC (n = 200 in each group) from MF (3.0 × 103 and 3.3 × 103 cells, respectively) than from SF (2.0 × 103 and 2.5 × 103 cells, respectively). Cumulus cells proliferated significantly in the presence of oestradiol, regardless of the concentration, during pre-incubation for 6 h (2.5 to 2.8 × 103 cells), as compared with the oestradiol-free controls (2.2 × 103 cells). These results demonstrate that the different abilities of cumulus expansion between COC (n = 200 in each group) from SF and MF may be due to the number of cumulus cells per COC. Pre-incubation in the presence of oestradiol stimulates the proliferation of cumulus cells and may improve the oocyte maturation of COC derived from SF.
271 NUCLEAR MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS - OOCYTE COMPLEXES
Authors: Cervantes M. P. , Anzar M. , Mapletoft R. J. , Palomino J. M. , Adams G. P.
Published: December 2012

Methods of producing wood bison embryos in vivo and in vitro are being developed in an effort to preserve the genetic diversity of this threatened species. Previous data from our laboratory suggest that oocytes collected 24 h after LH treatment had not yet achieved nuclear maturation. The objectives of this study were (1) to determine the optimal interval of time after hCG treatment required for in vivo maturation of cumulus–oocyte complexes (COC) in wood bison, and (2) to compare the maturational characteristics of COC after in vitro v. in vivo maturation. Follicular wave emergence was synchronized among bison cows (n = 25) by follicular ablation (Day –1) from May to June. Ovarian superstimulation was induced with FSH IM diluted in 5 mg mL–1 of hyaluronan (MAP-5, Bioniche, Belleville, Ontario, Canada) given on Day 0 (300 mg) and Day 2 (100 mg). Superstimulated cows were assigned randomly to 5 groups (n = 5/group): COC collected on Day 4 with no maturation (control), or matured in vitro for 24 or 30 h, or collected 24 or 30 h after treatment with 2000 IU of hCG IM on Day 4. The COC were collected by transvaginal ultrasound-guided follicle aspiration. In vitro maturation was done in TCM-199 with 5% calf serum, 5 µg mL–1 of LH, 0.5 µg mL–1 of FSH, and 0.05 µg mL–1 of gentamicin, at 38.5°C and in 5% CO2. To assess nuclear maturation, oocytes were stained with anti-lamin AC/DAPI (4′,6-diamidino-2-phenylindole). Nuclear stages were classified as germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Comparisons among groups were made by ANOVA and Fisher’s exact test (Table 1). A mean (± SEM) of 7.6 ± 0.6 COC was collected per bison; no differences were observed among groups (P = 0.37). Cumulus cell expansion was more extensive after in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30-h group (97%; P < 0.05). No COC were expanded in the control (0 h) group, and none reached MI. Maximal nuclear maturation was achieved in vitro by 24 h; that is, there was no difference in the proportion of MII-stage COC at 24 versus 30 h. However, between 24 and 30 h of in vivo maturation, the percentage of nuclear stages GV + GVBD decreased from 54 to 24% (P < 0.05), whereas nuclear stages MI + MII increased from 39 to 74% (P < 0.05). In conclusion, nuclear maturation occurred earlier in vitro versus in vivo, but the consequences of this difference are unknown. Although more than one-third of oocytes matured in vivo for 30 h were mature enough to permit immediate IVF, whether additional in vivo maturation time would be beneficial to fertilization rates remains to be tested.

Table 1.Nuclear status of wood bison oocytes after in vitro or in vivo maturation

Thanks to Bioniche Canada.
272 COMPARISON OF VIABILITY BETWEEN SEXED VERSUS CONVENTIONAL BEEF SEMEN AT 37°C and 5% CO2
Authors: Haarmann D. , Warshaw M. , Lorton S. P. , Kesler D. J. , Ferguson C. E.
Published: December 2012

This study was designed to compare the quality of sexed-sorted (X-bearing chromosome) semen and conventional semen over a 24-h period in a controlled in vitro system (37.5°C and 5% CO2). Both conventional and sexed semen (95% accurate for sexed) were collected from one Angus, Red Angus, and Hereford bull owned by CRI (Genex Cooperative, Inc., Shawano, WI, USA). The straws of sexed semen used had sperm cells sorted for X-bearing sperm (2 million sperm per straw) before cryopreservation. Semen samples were thawed in a warm water bath (37°C) and extended with BO-AB fertilization stock media. Samples were placed in the incubator for 15 min before evaluation for microscopic progressive motility, acrosomal integrity, and viability (Eosin B-Fast Green stain) at 0 (15 min after pre-incubation) 3, 6, 9, 15, and 24 h. Slides were evaluated on a heated microscope stage at 37.5°C at 400× magnification. Five replicates were performed over a 5-week period. One straw of conventional semen and one straw of sexed semen from each bull were used for each replicate. The percentage of motility, live/dead, slope of the regression line for percentage of motility, percentage of live with intact acrosome, and percentage of live for both sexed and conventional semen were analysed in SAS using the general linear models procedure (SAS Institute Inc., Cary, NC, USA) and a Tukey chi-squared post hoc test. There were no differences between bulls in each semen type; therefore, all bulls and all replicates were pooled for the final analysis. The number of live spermatozoa with an intact acrosome was higher (P < 0.05) for conventional semen compared with sexed semen at each evaluation time (Table 1). In addition, the motility remained higher (P < 0.05) for conventional semen than for sex-sorted semen throughout the experiment (Table 1). There was a difference (P < 0.05) between conventional and sexed semen in the proportion capable of fertilization (percentage of motile sperm × percentage of live sperm with an intact acrosome) from 0 h through 9 h post-thaw. The slope of the regression line for the decrease in motility plotted versus time was greater (P < 0.05) for conventional (–0.1140) than for sexed semen (–0.096); however, the slope of the regression lines for the decrease in the number of live or live with intact acrosomes was not different (P = 1.0) between conventional (–0.11; –0.11) and sexed (–0.11; –0.11) semen, respectively. These results indicate that sexed semen follows the same mortality pattern as conventional semen; however, at thawing it begins at a lower motility and viability.

Table 1.Comparison of sexed versus conventional semen in percentage of live with intact acrosomes and motility at different time points