bluuebunny/crossref_metadata_2025 huggingface dataset:
1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos
Authors: Duan J. E. , Jiang Z. , Alqahtani F. , Mandoiu I. , Dong H. , Zheng X. , Marjani S. L. , Chen J. , Tian X. C.
Published: December 2018

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.
10 Effect of 1 or 2 doses of prostaglandin in a resynchronization protocol for timed artificial insemination in beef cows
Authors: Pessoa G. A. , Martini A. P. , Baioco A. P. , Machado Filho E. F. , Pinto H. F. , Carloto G. W. , Sá Filho M. F. , Claro Junior I. , Alves Neto N.
Published: December 2018

The objective of this study was to compare the effects of 1 and 2 prostaglandin doses in a timed AI (TAI) resynchronization protocol on the pregnancy rate. This study was performed during the 2017-2018 breeding season on 10 commercial farms located in the central and southwest region of Rio Grande do Sul, Brazil. A total of 2347Bos taurus beef cows (Angus, Hereford) with a body condition score 2.90±0.15 (1=thin, 5=obese) and 40±5.1 days postpartum were used. In the beginning of first synchronization TAI protocol, on Day −11, the animals received 2mg of oestradiol benzoate IM (Gonadiol®, Zoetis Saúde Animal, São Paulo, SP, Brazil) and a progesterone (P4) intravaginal device (CIDR®, Zoetis). On Day −4, the cows received 12.5mg of dinoprost tromethamine IM (PGF, Lutalyse®, Zoetis). On Day −2, the animals received 1.0mg of oestradiol cypionate IM (ECP®, Zoetis) associated with the treatment with 300IU of eCG IM (Novormon®, Zoetis) and the P4 device was removed. The TAI was done on Day 0 (48 h after P4 device removal). The resynchronization protocol was started on Day 23 without pregnancy or oestrus detection; all cows were randomly assigned to receive a new, once-used, or twice-used P4 device (CIDR®) and given 2mg of oestradiol benzoate. On Day 30, pregnancy diagnosis was performed with transrectal ultrasonography. Pregnant cows (n=1021) had the P4 device withdrawn and received no further treatment. Nonpregnant cows (n=1326) were randomly assigned into 2 groups. The 1PGF group (n=674) received a single dose of PGF on Day 30, and the 2PGF group (n=652) received PGF on Days 30 and 32. On Day 32, all cows received eCG and ECP, and the P4 device was removed. In both groups the TAI was performed 48h after the P4 device removal (Day 34). Pregnancy diagnosis was performed by ultrasound 30 days after TAI (Day 64 of the breeding season). Data were analysed using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). The pregnancy rate obtained in the first synchronization protocol was 43.5% (1021/2347). The pregnancy rate in the resynchronization protocol was similar (P=0.49) between PGF doses [1PGF=39.5% (266/674); 2PGF=40.6% (265/652). No interactions were observed between P4 device use and PGF dose (P=0.38). The pregnancy rates were similar when comparing 1PGF and 2PGF using a new P4 device [1PGF=43.2% (35/81); 2PGF=56% (93/166); P=0.67], once-used P4 device [1PGF=39% (163/418); 2PGF=40.6% (140/345); P=0.64], or twice-used P4 device [1PGF=38.9% (68/175); 2PGF=22.7% (32/141); P=0.14]. In conclusion, a second dose of prostaglandin in the resynchronization protocol was not necessary to achieve high resynchronization pregnancy rates with TAI in Bos taurus beef cows.
118 A retrospective analysis of follicular dynamics, LH, estradiol-17β, and progesterone in prostaglandin F2α-induced estrus of Beetal goats
Authors: Murtaza A. , Khan M. I. R. , Ahmad W. , Sohail T. , Mohsin I. , Shahzad M. , Hussain M. , Tahir M. Z.
Published: December 2018

The objective of the study was to reveal the factors associated with variable interval to ovulation following a single dose of prostaglandin F2α (PGF2) in Beetal goats. In experiment 1, the extent of ovulation time was demonstrated after synchronizing Beetal goats (n=38) by using a single injection of PGF2 based on corpus luteum detection by B-mode transrectal ultrasonography. Ovulations were detected every 6h from PGF2 until ovulation. To explore the changes in follicular dynamics, endocrine profiles and behavioural events due to the variation in the interval to ovulation, another set of goats (n=13; experiment 2) were synchronized by a single dose of PGF2 given at random days of the luteal phase. Follicular dynamics were monitored every 12h from PGF2 until ovulation and oestrus was detected by apronized bucks every 6h from PGF2 treatment. Blood sampling (3mL) for oestradiol-17β and progesterone was done every 12h from PGF2 until ovulation and every 2h for LH assay by radioimmunoassay. Data were analysed by Student’s t-test, general linear models, and Pearson coefficient correlation (SPSS, version 20.0, SPSS Inc., Chicago, IL, USA). Results revealed that ovulation time varied from 60 to 96h after PGF2, and 57% of ovulations occurred by 72h after PGF2 (experiment 1). Based on this observation, goats (n=13) in second experiment were retrospectively divided into early and late ovulators (i.e. 72 and 84h following PGF2, respectively). Onset of oestrus, peak oestradiol-17β concentration, and LH surge after PGF2 were first observed in early than late ovulators (P<0.05). The goats having greater follicle diameter and smaller corpus luteum diameter at the time of PGF2 ovulated earlier than late ovulators (5.4±0.2v. 4.3±0.2mm and 10±0.6v. 11.8±0.3mm, respectively; P<0.05). Likewise, plasma progesterone concentration tended to be lower (P=0.087) in early than late ovulators. However, interval from onset of oestrus to peak LH and ovulation, duration of standing oestrus, interval from LH peak to ovulation, diameter and growth rate of preovulatory follicles, and number of ovulations were similar (P>0.05) between early and late ovulators. In conclusion, the size of dominant follicle and corpus luteum at the time of PGF2 determines the interval to ovulation following a single dose of PGF2 during the luteal phase.
14 Sperm membrane integrity in ejaculates from young bulls may be improved by single-layer centrifugation
Authors: Hurri E. , Johannisson A. , Lim-Verde I. , Morrell J. M.
Published: December 2018

Sperm quality in the first ejaculates of young bulls is often poor, leading to rejection of these samples for commercial freezing. It may be several weeks before sperm quality reaches the thresholds regarded as being acceptable for commercial production. Single-layer centrifugation (SLC) has been shown to separate robust bull spermatozoa from the rest of the ejaculate (Goodla et al. 2015 J. Dairy Sci. 97, 2204-2212; Nongbua et al. 2017 Reprod Domest. Anim. 52, 596-602). The aim of this study was to test whether SLC can be used to select spermatozoa from the ejaculates of young bulls and thus freeze samples earlier than is currently possible. Ejaculates (at least 3 per bull; n=33) were collected from 9 young Holstein or Swedish Red bulls, 255 to 415 days old. The sperm concentration was adjusted to 69×106 spermatozoa mL−1 with Andromed™ (Minitub, Tiefenbach, Germany) before layering part of the sample over 4mL of Bovicoll colloid. The remaining sample served as the unselected control. After centrifugation at 300×g for 20min, the sperm pellet was aspirated into fresh extender. Both control (uncentrifuged) and SLC samples were frozen and stored in LN pending analysis. Membrane integrity was evaluated by flow cytometry after staining for 10min at 37°C with SYBR14/propidium iodide (Live-Dead Sperm Viability Kit, L-7011; Invitrogen, Carlsbad, CA, USA). Paired t-test was used to compare results for control and SLC samples, and Pearson correlation was used for age and membrane integrity. The median age at which the samples were taken was 319 days (range: 255-415 days). Membrane integrity varied considerably among bulls (range: 11-72%). The mean proportion of membrane-intact spermatozoa in controls and SLC samples was 40±15 and 47±16%, respectively (P<0.01). In 21 of the 33 ejaculates (64%), the SLC sample had a higher membrane integrity than the controls (Table 1). Age in days was positively correlated with membrane integrity for SLC samples (r=0.40; P<0.05) but not for controls. These results suggest that SLC might be a useful technique for selecting membrane-intact spermatozoa from the ejaculates of young bulls. However, there is considerable variation among bulls, possibly due to age, which is currently being investigated. Other parameters of sperm quality, such as chromatin integrity, mitochondrial membrane potential reactive oxygen species production, and motility, are also being evaluated.

Table 1.Age and membrane integrity (mean±standard deviation) in ejaculates from young bulls before and after SLC (n=33)

This project was funded by the Royal Swedish Academy of Agriculture and Forestry, Viking Genetics, and the Faculty for Veterinary Medicine and Animal Health, SLU. We thank Viking Genetics for supplying the semen samples.
145 Inhibitory effects of gossypol on bovine in vitro embryo production
Authors: Hatamoto-Zervoudakis L. K. , Duarte Jr M. F. , Motheo T. F. , Tsuneda P. P. , Zervoudakis J. T.
Published: December 2018

Cottonseed and its derivatives are frequently used in cattle feed as an effective dietary fibre supply and high protein and energy food source. However, the cotton plant contains gossypol, which in its free form induces male and female infertility. Therefore, this study aimed to evaluate the effect of gossypol supplementation on bovine in vitro embryo production. Ovaries were retrieved from slaughterhouses, and cumulus-oocyte complexes (COC) were recovered by follicular puncture. Based on free gossypol concentration present on the in vitro maturation, sperm capacitation, IVF and in vitro culture media, grades I, II and III COC (n=646) were divided in 3 treatments: 0μg mL−1 (control), 5μg mL−1 (G5) and 10μg mL−1 (G10). The COC were matured under a humidified atmosphere of 5% CO2 in air at 38.5°C for 24h in 90-μL droplets containing TCM-199 supplemented with 10% FCS, 0.2mM sodium pyruvate, LH, FSH, 75μg mL−1 amikacin, 17β-oestradiol. Each droplet corresponded to one replicate (n=14) and contained 15 to 18 COC. Matured COC and sperm were co-incubated in droplets (8-13 COC per 90μL) of TALP-IVF media supplemented with 6mg mL−1 BSA, 0.2mM sodium pyruvate, 30μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, 75μg mL−1 amikacin under a 5% CO2 humidified atmosphere at 38.5°C, for 20h. For IVF, non-sexed frozen-thawed semen was selected with Percoll® gradient. The resulting pellet was subjectively evaluated for motility and concentration and then diluted to final concentration of sperm mL−1 with fertilization medium. Presumptive zygotes were then cultured in 90-μL droplets of SOFaaci medium supplemented with 2.7mM myo-inosytol, 0.2mM pyruvate, 2.5% FCS (v/v), 5mg mL−1 BSA, 75μg mL−1 amikacin, and maintained for 8 days at 38.5°C in a humidified atmosphere with 5% CO2 in air. Cleavage, blastocysts production and hatching rates were evaluated at Days 3, 7 and 8, respectively. Data were submitted to ANOVA for parametric data and Wilcoxon test for non-parametric variables using the SAS software (SAS Institute Inc., Cary, NC, USA). Significance level was set at 5%. Cleavage rates of the control (81.05%) and G5 (71.85%) were higher compared with G10 (19.64%; P < 0.0001). Blastocyst production was lower in G5 (12.18%) compared with control (30.35%), and the addition of 10μg mL−1 of free gossypol (G10) completely inhibited embryo development (0%; P < 0.0001). As for the percentage of hatched blastocysts, the control (66.75%) had greater values compared with G5 (34.52%; P < 0.0001). Thus, the addition of 5 and 10μg mL−1 of free gossypol are extremely hazardous for in vitro bovine embryo development. Whether these deleterious effects take place in a similar fashion during in vivo embryo production remains to be investigated.
148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)
Authors: Uchuari M. L. , Artica M. , Villanueva J. C. , Huanca W. F. , Huanca W.
Published: December 2018

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles >2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas.

Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine

Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.
152 Correlation between testicular and accessory sex glands biometric characteristics in Nellore and Caracu bulls
Authors: Rodrigues N. N. , Vrisman D. P. , Rossi G. F. , Freitas A. P. , Zorzetto M. F. , Souza L. L. , Taira A. R. , Vicente W. R. R. , Monteiro F. M. , Oliveira M. E. F.
Published: December 2018

The objective of this study was to determine the correlation between testicular and accessory sex gland measurements in Nellore (Bos taurus indicus) and Caracu (Bos taurus taurus) bulls. Bulls (n=282, including 203 Nellore from 12 to 61 months and 79 Caracu from 12 to 48 months) had their reproductive organs measured. Scrotal circumference was measured with a tape. Length (dorso-ventral) and diameter (mid-lateral) of testes were measured using a graduated ruler. Testicular volume (V) was calculated by the cylinder formula: V=2[(R2)×π×L], where R=radius (diameter/2), L=testicular length and π=3.14 (Fields et al. 1979 J. Anim. Sci. 48, 1299-1304). B-mode ultrasonographic examinations with a 7.5-MHz transrectal linear-array transducer were performed to obtain the mean of 3 vertical dimensions of the vesicular glands, disseminated prostate, ampulla of vas deferens, and lumen of ampulla, and cranio-caudal and dorso-ventral dimensions of the prostate body and bulbourethral glands. For paired organs, the mean was calculated and used in analyses. Biometry data of testes and accessory sex glands were analysed by the PROC CORR (Pearson correlation) of the SAS program (SAS Institute Inc., Cary, NC, USA; P<0.05). Testicular measurements (scrotal circumference, diameter and length) were positively correlated with each other (r=0.69 to 0.92). Similarly, biometrics of the accessory sex glands had positive correlations with testes. The vesicular glands had correlations of r=0.62, 0.43, 0.58 and 0.59 with testes length, diameter, and volume and scrotal circumference, respectively. Correlations testicular biometry characteristics and ampulla of vas deferens ranged from 0.52 to 0.68, whereas those between testicular characteristics and lumen of ampulla were much lower (r=0.28 to 0.37), perhaps due to bulls riding penmates and ejaculating before the ultrasonographic examination. The dorso-ventral measure of the prostate body had positive correlations with size of testes (r=0.13 to 0.28), whereas cranio-caudal measurement of this gland was not correlated with size of testes. Finally, there were positive correlations between testes and disseminated prostate (0.28 to 0.36), and testes and bulbourethral glands, both in the dorso-ventral and cranio-caudal dimensions (0.17 to 0.42). In conclusion, testicular biometry in bulls was correlated with measurements of accessory sex glands, perhaps due to testosterone synthesis, which is essential for accessory sex gland development.
159 Effect of bisphenol A and bisphenol S on AMH and AMHR mRNA expression during in vitro bovine oocyte maturation and early embryo development
Authors: Saleh A. , Favetta L.
Published: December 2018

Exposure to chemicals with known endocrine-disrupting effects, such as bisphenol A (BPA) and bisphenol S (BPS), leads to repercussions on oocyte development and, ultimately, on fertility. Bisphenol A is a plasticizer used worldwide that has been detected in blood, urine, tissue samples and follicular fluid. Due to its widely reported detrimental effects, BPA has been substituted with its analogue BPS. Previous experiments in our laboratory have shown that exposure of bovine oocytes to physiologically relevant doses of BPA resulted in spindle abnormalities, reduced meiosis progression, decreased blastocyst rate and gene expression changes. However, the effects of BPS have not yet been investigated. Anti-Müllerian hormone (AMH) has been reported to be a good marker of ovarian reserve and oocyte developmental capability and is commonly used in assisted reproduction for diagnostic measurements. There is evidence that women undergoing IVF with higher BPA levels have lower AMH levels and pregnancy success. The aim of this study was to assess the effect of BPA and BPS on AMH and its receptor as measures of oocyte developmental capability. Abattoir-derived bovine cumulus-oocyte complexes (COC) were matured in vitro in 4 groups: (1) control, (2) vehicle (0.1% ethanol), (3) BPA (0.05mg mL−1 in 0.1% ethanol), and (4) BPS (0.05mg mL−1 in 0.1% ethanol). Pools of 30 COC, 30 denuded oocytes, and cumulus cells corresponding to denuded oocytes were collected for each of the 4 experimental groups, and a minimum of 4 biological replicates were used for each analysis. Anti-Müllerian hormone and AMH receptor mRNA expression was measured in COC, denuded oocytes, and their corresponding cumulus cells using quantitative real-time PCR. Statistical analyses were performed using 1-way ANOVA. Results showed a decrease (P<0.05) in AMH mRNA expression in BPA-treated oocytes (without cumulus cells). In addition, there was an increase (P<0.05) in mRNA AMH receptor levels in COC when treated with BPS. Finally, analyses on cumulus cells alone showed an increase (P<0.05) in the AMH receptor mRNA levels in BPA-treated cells. These results suggest that BPA has an effect on AMH mRNA transcript levels in oocytes, while affecting the receptor expression in cumulus cells. Conversely, BPS affects AMH indirectly, increasing the mRNA levels of its receptor only. Further investigation of the effects of BPA and BPS on AMH expression in in vitro-produced blastocysts derived from treated oocytes will aid in understanding the potential consequences of exposure to BPA and its analogues on early embryonic development.
164 Evaluation of the lipid content of in vitro-matured bovine cumulus - oocyte complexes in the presence of natriuretic peptides A, B, and C types
Authors: Schefer L. , Schwarz K. R. L. , Fernandes H. , Paschoal D. M. P. , Castro F. C. C. , Leal C. L. V.
Published: December 2018

One of the difficulties still observed in in vitro production (IVP) of bovine embryos is the lower cryotolerance of such embryos, which has been related to their increased lipid accumulation during culture in the presence of fetal bovine serum (FBS). Previous studies have indicated that the cyclic guanosine monophosphate (cGMP) pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). Synthesis of cGMP can be caused by activation of membrane guanylate cyclase (mGC), also called natriuretic peptide receptors (NPR1 and NPR2), which are activated by natriuretic peptides (NP) A, B, and C types (NPPA, NPPB, and NPPC). The objective of this study was to investigate the influence of supplementation with NP during in vitro maturation (IVM) on lipid content and nuclear maturation of bovine COC. Pools of 25 COC were submitted to IVM in TCM-199 with 0.2mM sodium pyruvate, 10μg mL−1 gentamicide, 0.5μg mL−1 FSH, 10% FBS, and NPPA (10−5 M), NPPB (10−7 M), or NPPC (10−5 M). The control group was matured without NP. After 24h, cumulus cells (CC) were removed and oocytes (OO) were fixed and permeabilized in 4% paraformaldehyde+0.5% Triton for 20min, stained with 10μg mL−1 Hoechst 33342 for 15min and 1μg mL−1 Nile Red for 30min. Then, the OO were placed in 13μL of ProLong (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, covered with a coverslip, and submitted to epifluorescence microscopy to evaluate nuclear maturation (emission 445-450nm and excitation 475-490nm) and lipid content (emission 590nm and excitation 516-560nm). Data for 4 replicates/group were tested for normality of results and homogeneity of variance, and then submitted to ANOVA, followed by Tukey test using a GraphPad Prism software (GraphPad Inc., San Diego, CA, USA), with a significance level of 5%. Nuclear maturation was influenced only by one of the NP added to IVM medium, NPPC, which reduced maturation rate (68.3% metaphase II, MII) compared with the control (82.7% MII; P<0.05). Maturation rates of NPPA (79.5% MII) and NPPB (85.1% MII) did not differ from the control (P>0.05). Activation of mGC by NPPB generated oocytes with lower lipid content (34.02±1.2 IF/μm2) compared with control (36.98±0.7 IF/μm2; P<0.05). Neither NPPA (36.5±1.4 IF/μm2) nor NPPC (39.3±1.4 IF/μm2) was able to reduce the lipid content in oocytes matured in vitro relative to control (P>0.05). Different NP may have different effects on maturation and lipid content in in vitro matured bovine oocytes; NPPB may be favourable for reducing the lipid content of matured bovine oocytes in vitro.
168 Melatonin slightly alleviates the effect of heat shock on nuclear maturation of the bovine oocyte
Authors: Shimoni C. , Kalo D. , Hansen P. J. , Roth Z.
Published: December 2018

Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means±s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7v. 55%, respectively; chi-squared, P<0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32v. 13.7%, respectively; chi-squared, P<0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8±5.32v. 33.9±4.08 pixels intensity, respectively; P<0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0±4.78; P<0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.
18 Characterisation of early embryonic cellular defects after somatic cell nuclear transfer in fish
Authors: Rouillon C. , Depincé A. , Chenais N. , Le Bail P.-Y. , Labbé C.
Published: December 2018

Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n=16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n=69/95 clones v. 96%, n=96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n=14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n=7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.
180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes
Authors: Amoroso-Sanches F. , Gonzalez-Castro R. , Stokes J. , Carnevale E.
Published: December 2018

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (> 70%), DNA integrity (<8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P<0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P<0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.
187 Phenotype switch of human fibroblasts into trophoblastic cells
Authors: Arcuri S. , Manzoni E. F. M. , Gandolfi F. , Brevini T. A. L.
Published: December 2018

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is crucial for implantation of the blastocyst and gives rise to specialised populations of trophoblast cells in the definitive placenta. However, our understanding of these early differentiation events is limited, particularly in humans, because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Here we describe experiments aimed at converting human fibroblasts into trophoblastic cells, using 5-azacytidine (5-aza-CR) to erase the original phenotype and a cocktail containing bone morphogenic protein 4 (BMP4) and inhibitors of the activin/nodal/ERK signalling pathways, to drive trophoblastic differentiation. The method required 3 main steps: (1) preparation of mouse embryonic fibroblasts (MEF) monolayer, and culture and collection of MEF conditioned medium (CM); (2) culture of human fibroblasts, obtained from a skin biopsy, and epigenetic erasure with 5-aza-CR for 18 h; (3) use of CM, with BMP4 (50 ng mL−1), PD0325901 (1 µM), CHIR99021 (1 µM), and PD173074 (0.1 µM) to differentiate erased human fibroblasts to trophoblast cells. Morphology changes were monitored along the process. In the initial phase of differentiation, a mixture of cell types appeared, including small cells growing in clusters and giant cells possessing very large nuclei. After 2 weeks of culture, cells displayed a distinct trophoblast-like morphology and showed the presence of typical clustering/lacunae monolayer patterning. Cells continued to proliferate and maintained a normal karyotype. BMP4-mediated differentiation was also assessed by quantitative RT-PCR using primers specific for 2 trophoblast markers: keratin 7 (KRT7) and caudal type homeobox 2 (CDX2), that are absent in the original fibroblasts. Their expression appeared by Day 3 of induction and was strong and steady throughout the process, confirming that the acquisition of a trophoblast-like morphology was supported by the activation of trophoblast-specific gene transcription. The results demonstrate the possibility of obtaining trophoblast-like cells through the conversion of human fibroblasts and confirm the involvement of BMP4-together with the inhibition of the activin/nodal/ERK signalling pathway-to activate early trophoblast differentiation in vitro. The challenge for future experiments will be to determine the precise role of BMP signals in human trophoblast definition in vivo, which, to our knowledge, has not been elucidated yet. The method here described is efficient and reproducible and has the advantage of utilising easily accessible cells as a starting population. It can be used for a variety of applications, including drug discovery and stem cell research, as well as to implement studies on the pathogenesis of developmental disorders with trophoblast defects.
This research was supported by Carraresi Foundation. Authors are members of the COST Actions CA16119 and CM1406.
203 Newly designed liposome nanoparticles for drug delivery into boar spermatozoa
Authors: Feugang J. M. , Eggert M. W. , Park S. B. , Popoola M. A. , Steadman C. S. , Arnold R. R. , Ryan P. L. , Willard S. T.
Published: December 2018

Liposomes are spherical vesicles with a membrane composed of at least one phospholipid bilayer. They have been successfully applied for intracellular drug or genetic material delivery in many biomedical areas. In agriculture, however, their use to produce transgenic farm animals through exogenous DNA transfection of spermatozoa remains unsatisfactory. Here, we tested a newly designed liposome preparation for effective and harmless interactions with boar spermatozoa. Extended fresh boar semen samples were gently centrifuged (N=3 independent replicates), and sperm pellets were resuspended in PBS. Sperm concentrations were then adjusted to 2×108 mL−1 and aliquoted in 0.5mL for labelling with various doses of fluorescent liposomes (0, 15, 30, or 60µg), for plasma membrane labelling (Experiment 1) or with nonfluorescent liposomes loaded with doxorubicin (0 or 60µg) having natural fluorescence property, for nucleus labelling (Experiment 2). After co-incubation for 45 to 60min at 37°C, sperm aliquots of each treatment were immediately evaluated for motility and morphology characteristics using a computer-assisted sperm analyzer. Remaining sperm-liposome mixtures were centrifuged to remove the excess of liposomes, and pelleted spermatozoa were imaged with the In vivo Imaging System (IVIS: PerkinElmer, Waltham, MA, USA) and fluorescence confocal laser microscope (LSCM; Zeiss, Oberkochen, Germany). Data were statistically analysed using one-way ANOVA or Student’s t-test, with P<0.05 indicating a significant difference. In Experiment 1, the proportions of motile and forward progressive spermatozoa were significantly (P<0.05) increased by the presence of 30µg (65±4% and 46±3%, respectively) and 60µg (66±1% and 48±1%, respectively) of liposomes compared with 0µg, the control (51±3% and 36±3%, respectively). In Experiment 2, doxorubicin-loaded liposomes significantly (P<0.05) improved the proportions of motile and progressive spermatozoa (87±2% and 69±8% v. 71±8% and 55±8% for the control, respectively). The percentages of static and abnormal spermatozoa (bent tail or distal cytoplasmic droplet) were significantly decreased by the presence of liposomes in both experiments (P<0.05). In contrast, the velocity parameters of spermatozoa were not significantly affected by the presence of liposomes (P>0.05). The microscope imaging revealed sperm-liposome interactions with displayed sperm membrane and nucleus fluorescence in Experiments 1 and 2, respectively. The proportion of stained cells in each experiment is in evaluation by flow cytometry. In conclusion, the newly designed liposomes were effective for simple interactions with the plasma membrane and molecule delivery to boar spermatozoa, while improving sperm motility. Further studies are ongoing with the replacement of doxorubicin with a DNA fragment.
This work was supported by USDA-ARS Biophotonics Initiative #58-6402-3-018.
206 Towards the generation of transchromosomic goats for the production of fully human immunoglobulin
Authors: Fan Z. , Regouski M. , Brandsrud M. , Wu H. , Liu Y. , Wang Z. , Sullivan E. , Polejaeva I. A.
Published: December 2018

We have previously reported that inactivation of endogenous immunoglobulin (Ig) genes can enhance the production of human polyclonal antibodies (hpAb) in transchromosomic (Tc) cattle containing a human artificial chromosome (HAC) comprising the entire human Ig gene repertoire. Goats offer the advantages of having a shorter gestation period and growing to adult size much faster than larger ungulates. Therefore, based upon our previously established Tc cattle platform, in this study, we aimed to generate immunoglobulin lambda light chain knockout (IGLλ

−/−
)/HAC Tc goats expressing hpAb using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. First, we designed multiple specific single-guide RNAs (sgRNA) targeting the coding sequences of constant regions of goat IGLλ1, IGLλ2 (both in NC_030824.1), and IGLλ3 (NW_017189530.1) and constructed corresponding gene targeting vectors. Gene targeting efficiency analysis in goat fibroblasts showed that the sgRNA were efficient in directing Cas9 to generate targeted indels in corresponding IGLλ genes with efficiencies ranging between 20 and 30%. We then targeted the IGLλ genes and generated a cloned IGLλ

−/−
fetus by SCNT (first round of cloning; G1) using single cell-derived IGLλ

−/−
fibroblast colonies as nuclear donors and established IGLλ

−/−
fetal fibroblast cell lines. Second, we established IGLλ

−/−
/HAC fibroblast colonies by transferring the isKcHACΔ, a HAC containing the entire unrearranged human immunoglobulin heavy-chain and kappa-chain sequences, into the IGLλ

−/−
fetal fibroblast cells via microcell-mediated chromosome transfer (MMCT; Matsushita et al. 2014 PLoS One 9, e90383). The resulting IGLλ

−/−
/HAC fibroblasts were used as nuclear donors for the production of IGLλ

−/−
/HAC goats by SCNT (second round of cloning; G2). A total of 311 one-cell stage G2 embryos were generated using 4 IGLλ

−/−
/HAC fetal fibroblast colonies and surgically transferred into 19 oestrus-synchronized recipients. Seven pregnancies were confirmed (7/19; 36.8%) with ultrasonography at Day 60±3 of gestation, and 1 pregnancy (1/7; 14.3%) developed to term, leading to the birth of a live and healthy kid. Genetic and serological characterisation of this Tc goat produced by the second round of SCNT is in progress.
208 The effect of copper on the differentiation of adipose-derived stem cells into osteoblasts
Authors: Bane T. , Siegel L. , Bertels J. , Ratz K. , Rubessa M. , Wheeler M.
Published: December 2018

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate whether osteogenic medium infused with copper influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Copper is a key cofactor for lysyl oxidase, an enzyme involved in producing a collagen matrix through which bone can grow. Lysyl oxidase expression is up-regulated in bone marrow stromal cells (Khosravi et al. 2014 PLoS One 9, e100669). Our hypothesis was that the presence of copper in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 7 different treatments: 5 concentrations of copper in the osteogenic medium (0.1, 1, 10, 50, and 100 µM) plus 2 control treatments (osteogenic medium without copper and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa stains. In each well, we counted the total number of nodules that were either formed or forming. Data were analysed using the generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Armonk, NY). The least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that more nodules were formed in the 0.1 and 1 µM copper groups compared with the osteogenic control, but there was no statistical difference between those 2 treatments. Table 1 illustrates the total number of formed and forming nodules in addition to their standard deviation. There was a positive effect on nodule formation when copper concentrations of 0.1 and 1 µM were added to the osteogenic medium. In contrast, copper concentrations of 50 and 100 µM had a cytotoxic effect. These results confirm that low concentrations of copper have a positive effect on osteogenesis. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with copper incorporated into their matrix.

Table 1.The average number (standard deviations in parentheses) of total formed and forming osteoblast nodules compared between treatment groups
21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen
Authors: Tshabalala M. M. , Nephawe K. A. , Mphaphathi M. L. , Pilane C. M. , Nedambale T. L.
Published: December 2018

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P<0.05. Sperm motility and membrane integrity were significantly higher (P<0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P<0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.