bluuebunny/crossref_metadata_2025 huggingface dataset:
174 Follicular fluid extracellular vesicles obtained from Holstein cows kept under thermoneutral or heat stress conditions modify gene expression of in vitro-matured oocytes
Authors: Dalanezi F. M. , Ferrazza R. A. , Ochoa J. C. , Mogllón H. D. , Destro F. C. , Franchi F. F. , Fontes P. K. , Castilho A. C. S. , Schmidt E. M. S. , Sartori R. , Ferreira J. C. P.
Published: December 2018

Heat stress (HS) has a massive impact on bovine reproduction. In cows, some of these deleterious effects involve follicular development and oocyte quality. Extracellular vesicles (EV) secreted by granulosa cells play a critical role in the intrafollicular environment by directly influencing cumulus cells and oocyte functions. The objective of this study was to investigate the effect of follicular fluid EV obtained from Holstein cows kept under thermoneutral (TN) or HS conditions, on in vitro bovine oocyte maturation. Non-lactating Holstein cows were synchronized with the Ovsynch protocol and also received an intravaginal progesterone device. From ovulation day (Day 1), cows were randomly assigned to TN (26°C, 73% humidity; n=12) or HS (36°C, 70% humidity; n=12) environments. On Day 9, 2 follicles (F1 and F2) were individually aspirated and all remained follicles were ablated. Then, on Day 14, newly formed F1 and F2 were also aspirated. Follicular fluid from all follicles from each treatment was pooled and EV were obtained according to Silveira et al. (2017 PLoS One 12, 1-25) and diluted in PBS (100μL of PBS per mL of follicular fluid centrifuged). Pools of 20 cumulus-oocyte complexes (COC) grade 1 or 2 (Stojkovic et al. Biol Reprod 200164, 904-992], predominantly from Bos indicus, were submitted to the following treatment groups: Control (n=4): matured in 90μL of TCM-199 with Eagles’ salts, glutamine, NaHCO3, pyruvate, amikacin, and FSH (base medium); TN (n=4): matured in 81μL of base medium+9μL of TN EV suspension; and HS (n=4): matured in 81μL+9μL of HS EV suspension. All treatments were carried out at 38.5°C for 24h in a humid atmosphere with 5% CO2. After 24h of maturation, COC were evaluated for meiotic progression (Hoechst 33342 stain), DNA integrity (TUNEL stain), and expression of genes related to oocyte quality (TaqMan assay, Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Results were analysed using ANOVA followed by Tukey post hoc test (P<0.05). When the experimental groups were compared with the control group, there was no treatment effect on meiotic progression, DNA integrity, or gene expression of cumulus cells. In the oocytes of the TN group, the genes HSF1, IGFBP2, BMP15, GDF9, CDCA8, HAS2, RPL15, STAT3,and PFKP were less expressed. We concluded that oocytes matured in the presence of EV from follicular fluid of cows kept under TN conditions had lesser expression of genes related to oocyte quality.
This study was supported by FAPESP (Grant #2012/18297-7) and CAPES Foundation of Brazil.
176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development
Authors: Honkawa Y. , Gen Y. , Hyon S.-H. , Kubota C.
Published: December 2018

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value<0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.
177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes
Authors: Keim J. , Liu Y. , Polejaeva I.
Published: December 2018

In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P<0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P>0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P<0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate.
Supported by RFBR (18-29-07089) and UAES (1343).
179 Decondensation of bovine spermatozoa after dithiothreitol treatment
Authors: Gatenby L. , Bondioli K. R.
Published: December 2018

Dithiothreitol (DTT) has been shown to reduce protamine disulfide bonds present in the outer acrosomal membrane of sperm. These disulfide bonds provide bull sperm with greater membrane stability and an increased resistance to decondensation compared with other species. (Hutchinson et al. 2017 Biophysical J. 113, 1925-1933). This can result in asynchronous fertilization due to the sperm nucleus failing to properly decondense and form male pronuclei after assisted reproductive techniques, such as IVF or intracytoplasmic sperm injection (ICSI) (Águila et al. 2017 Reproduction 154, 307-318). By reducing these bonds and disrupting the acrosomal membrane, DTT can minimize this complication, allowing the sperm nucleus to decondense more readily. This study aimed to assess bovine sperm, treated or not with DTT, for changes in morphology and to track the progress of decondensing sperm nuclei after treatment. Frozen-thawed bovine semen, utilising only egg yolk extender, was prepared by the swim-up method in the presence or absence of DTT at 5mM. After a 1-h incubation time, permitting sperm to swim up, sperm in the top 0.5mL in each culture tube were washed by centrifugation and observed hourly. Changes in morphology of DTT-treated and nontreated sperm were observed through a combination of phase-contrast and Hoffman modulation contrast microscopy. To assess DNA dispersal, DTT-treated sperm were stained with Hoechst and viewed under fluorescence microscopy. The DTT-treated sperm exhibited time-dependent changes in morphology, including altered movement, head folding at the equatorial segment, bending and swelling of the mid-piece, and expansion of the sperm head accompanied by a cratered appearance, signalling a partially decondensed nucleus, which progresses to full decondensation, and last, complete dispersal of DNA. These changes were seen over a 7-h incubation period in HEPES-TALP. No such changes were observed in nontreated sperm. The proportion of sperm in each stage of decondensation was estimated by a single observer at hourly time points in at least 8 repetitions. Mean estimated proportions of each stage of decondensation over time was analysed through factorial ANOVA and found significant (P<0.001). An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the third hour and increased to 62% by the fifth hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at 5h to 50% at 7h. A remaining 10% of sperm remained unaffected and showed no changes to morphology. These results were found to be in agreement with other studies and indicate that 5mM DTT treatment induces decondensation of bovine sperm nuclei, which may assist in male pronuclei formation for other assisted reproductive technologies.
183 Generation of porcine embryonic stem cell lines derived from nuclear transfer embryos reconstructed with induced pluripotent stem cells
Authors: Haraguchi S. , Dang-Nguyen T. Q. , Wells D. , Fuchimoto D. , Fukuda T. , Tokunaga T.
Published: December 2018

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.
184 Effect of selenium on the differentiation of porcine adipose-derived stem cells into osteoblasts
Authors: Siegel L. , Bane T. , Bertels J. , Ratz K. , Rubessa M. , Wheeler M.
Published: December 2018

Bone damage is a common occurrence and although traditional bone healing methods work well, they are not always able to fix all issues pertaining to the injury. However, recent research on stem cell therapies using adipose-derived stem cells (ASC) has shown that ASC that differentiate into osteoblasts can be used as a potentially better solution for bone injuries. Selenium is a nutritional trace element that has been found to be essential in the production of selenoproteins and bone growth. Selenium plays an integral role in osteoblast cell differentiation and proliferation. The aim of this experiment was to test whether adding selenium to osteogenic influences the differentiation potential of ASC into osteoblasts. Porcine ASC were isolated as described (Monaco et al. 2009, Open Tissue Eng. Regen. Med. J. 2, 20-33). Seven different treatments were given to the cells: a negative control of DMEM, a positive control of osteogenic medium, and 5 concentrations of selenium in the osteogenic medium (10, 5, 1, 0.5, and 0.1 µM). Medium was changed twice a week for 4 weeks, at the end of which the cells were stained with Alizarin Red S stain. The number of osteoblastic nodules in each well were counted and divided into the categories of “forming” and “formed.” A generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Chicago, IL, USA) was used to analyse the data. The least statistical difference (l.s.d.) post hoc test was used to perform statistical multiple comparison with an α-level of 0.05. The results showed (Table 1) that medium containing selenium concentrations of 0.1, 0.5, and 1.0 µM were able to develop more nodules than the osteogenic medium. There was no statistical difference in the total amount of forming and formed nodules in those 4 groups; however, there was a statistical tendency for a difference (P=0.06) between the medium with 1 µM selenium and the osteogenic medium. Medium containing 10 or 5 µM selenium had lower nodule counts (both forming and formed) than osteogenic medium. There was no statistical difference between 10 µM and DMEM. These results suggest that low concentrations of selenium have a positive effect on nodule formation, whereas higher concentrations are detrimental.

Table 1.The average number (standard deviations in parentheses) of formed, forming, and total osteoblastic nodules in each of the different treatments
186 The effects of different concentrations of MgSO4 in osteogenic differentiation
Authors: Padoveze L. R. , Rubessa M. , Ambrosio C. E. , Wheeler M. B.
Published: December 2018

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.
19 Success of handmade cloning in a commercial setting
Authors: Waybright T. , Sonsteby S. , Vajta G.
Published: December 2018

The purpose of this field trial was to determine whether handmade cloning could be used in a commercial setting to produce, transport, and implant embryos into recipients and to determine blastocyst and pregnancy rates. Donor animals and recipients were housed on 2 commercial farms, Farm A and Farm B. Ear notches were collected, grown in DMEM 10% FCS and 1% penicillin-streptomycin, and incubated at 38°C. Ovaries from a local abattoir were processed to collect cumulus-oocyte complexes for cloning. The cloning process included the following: (1) addition of demecolcine to maturation media at 22h, (2) bisection at 24 to 26h, (3) fusion at 25 to 27h, (4) activation at 30h, and (5) culture at 36h. After maturation, the cumulus cells were removed from the oocytes by incubating in 0.1% (wt/vol) hyaluronidase in HEPES-buffered TCM-199 with 2% (vol/vol) steer serum (T2) for 5min, followed by vortexing for 3min. The resulting cumulus-free oocytes were incubated in maturation media containing 0.5µg mL−1 of demecolcine for 2h. Next, the zona pellucida was removed with 0.2% (wt/vol) pronase in T2. An ultrasharp cutting blade was used to bisect the oocytes under a stereomicroscope, producing karyoplasts containing extrusion cones and cytoplasts. Fusion of 2 cytoplasts with a fibroblast was performed on a BTX fusion slide (San Diego, CA, USA) using a single direct current pulse of 100V for 9 µs. After fusion, the reconstructed embryos (REC) were incubated in SOFaaci for 3h until activation. The REC were activated with 10µM calcium ionophore for 5min in T2, followed by incubation in SOFaaci containing 2mM DMAP for 6h. Activated REC were individually cultured in well-of-the-wells (Vajta et al. 2000Mol. Reprod. Dev. 55, 256-264) containing SOFaaci without serum in 6% CO2, 5% O2, and 89% N2 for 7 days. For transport, 2-mL transfer tubes were filled with 400 uL of SOFaaci; overlayed with oil; gassed with 6% CO2, 5% O2, and 89% N2; loaded with 1 embryo per tube; and placed into a 39°C portable incubator. On Farm A, 34 REC were produced, with 13 developing to blastocyst stage (38% blastocyst rate). After a 1.5-h transport, 7 grade 1 expanded blastocysts were implanted into 7 synchronized recipients. At the 90-day pregnancy check, 3/7 (42%) were pregnant. On Farm B, 35 REC were produced, with 14 grade 1 morulas or early blastocysts developing (40% blastocyst rate). After a 6-h transport, 9 morulas or early blastocysts were implanted into 9 synchronized recipients. At the 90-day pregnancy check, 2/9 (22%) were pregnant. Overall, 5/16 (31%) of recipients remained pregnant by month 8 of gestation. In conclusion, handmade cloning is a practicable method to produce, transport, and implant embryos into recipients in a commercial setting.
191 The effect of high and low doses of follicle-stimulating hormone on embryo collection in Romanov sheep
Authors: Carwell B. , Carwell D. , Hubbard J. , Stuerman D.
Published: December 2018

The use of embryo transfer in small ruminants has served as a way of increasing superior female genetics. This technique, although not new, has been adopted by an increasing number of breeders over the past few years. The Romanov sheep breed is considered a natural litter-bearing breed compared with other small ruminant species, which generally have only 1 or 2 offspring during a natural parturition. This experiment was designed to evaluate the effect of FSH dosage on embryo production and collection in Romanov sheep. Sheep donors (n=12), with an average age of 2±1.4 years and an average body weight of 43±6.7kg, were randomly assigned to either a high (175mg) or low (140mg) dosage treatment of FSH during the breeding season. Hormone injections were given in a decreasing dosage twice daily over a period of 5 days. Donors were naturally cycling before being subjected to a CIDR-G protocol for a period of 15 days, with hormone injections beginning on Day 11. Donors were mated with rams (n=2) of known fertility for a total of 3 hand matings. After the third hand mating, the ram was turned into the breeding pen with the ewes. Embryos were collected 7 days after breeding and evaluated for transfer. A one-way ANOVA was utilised to analyse statistical differences between groups with significance set at (0.05). Results are given as mean±s.e.m. Only embryos of quality grades 1 and 2 were transferred during this study. No differences were detected between the 2 groups for total embryos collected between both high (7.6±6) and low (6.6±5) FSH groups. Furthermore, no differences were detected between both high and low FSH groups for the number of embryos transferred (5.6±5 and 6.3±4, respectively). The results of this study suggest that a lower total dosage (140mg) of FSH can be used in superovulation programs of Romanov ewes without affecting the total number of embryos collected and transferred. However, further investigation should be performed with various levels of FSH to determine the most accurate dosage for Romanov sheep.
192 The use of human chorionic gonadotropin seven days after synchronized estrus for the increase of luteal tissue in Morada Nova ewes
Authors: Vergani G. B. , Trevizan J. T. , Pereira V. S. A. , Fonseca J. F. , Garcia A. R. , Esteves S. N. , Oliveira M. E. F.
Published: December 2018

The study was performed to evaluate whether the use of hCG 7 days after synchronized oestrus in Morada Nova ewes would increase the number and size of corpora lutea. Multiparous Morada Nova ewes (n=115, mean weight of 36.6kg, body condition score 3.13 on 1 to 5 scale) were used. Oestrus was synchronized by using an intravaginal sponge impregnated with medroxyprogesterone acetate (60mg, Progespon®, Zoetis, Parsippany, NJ, USA) for 6 days and eCG (200IU, IM Novormon®, Zoetis) plus PGF2α analogue (37.5μg, D-cloprostenol, IM, Vetglan®, Hertape Calier, Spain), both administered 24h before the sponge removal. Seven days after the synchronized oestrus, hCG (300IU, IM, Vetecor®, Hertape Calier; n=57) or normal saline (control group, 1mL, IM, 0.9% NaCl, Eurofarma Laboratory SA, Sao Paulo, Brazil; n=58) was injected. B-mode ultrasound examinations of the ovaries were performed using the equipment MyLab Vet 30 Gold (Esoate, Genoa, Italy) on Day 7 (corresponding to the day of hCG or physiological solution administrations), and 6 days later (Day 13). The number of corpora lutea present and the size of luteal tissue (sum of diameter and area) were determined. Data were analysed by ANOVA with Tukey’s post hoc test (mean±s.e.m.) using SAS software (SAS Institute Inc., Cary, NC, USA). On Day 7, the number of corpora lutea was similar (P>0.05) between the hCG and control groups (1.58±0.09v. 1.57±0.08, respectively); however, the number was higher on Day 13 (P<0.05) in the hCG group (2.65±0.13) than in the control group (1.69±0.07). Similarly, there was no difference (P>0.05) between groups in diameter (16.9±0.82v. 16.0±0.71mm) and area (1.61±0.07v. 1.45±0.06cm2) for hCG and control groups, respectively, on Day 7. Differences were observed on Day 13, such that the hCG ewes had luteal tissue of larger (P<0.0001) diameters (27.13±1.25v. 18.44±0.85mm) and area (2.81±0.13v. 1.71±0.08cm2) than ewes of the control group. In conclusion, 300IU of hCG injected 7 days after synchronized oestrus is effective to increase the number of corpora lutea and in the size of luteal tissue. Despite indications of a favourable effect on luteal dynamics, the effects on progesterone production and conception rate need to be examined.
This study was supported by CNPq and EMBRAPA (process no. 02.13.06.026.00.02).
193 Melatonin implants in spring improve embryo production of aged ewes after superovulation regardless of endometrial progesterone receptor expression
Authors: Abecia J. A. , Meikle A. , Vázquez M. I. , Casao A. , Forcada F. , Sosa C.
Published: December 2018

Twenty-three Rasa Aragonesa aged ewes (average age: 10.3±0.3 years) were used to determine the effect of melatonin on ovulatory response, embryo production, and endometrial expression of progesterone receptors (PR) after superovulation. Ewes were treated (M, n=13) or not (control, C, n=10) with melatonin implants in March (Day 0, Northern Hemisphere autumn), and received intravaginal progestogen sponges for 14 days on Day 77. Superovulatory treatments consisted of 8 doses in decreasing concentrations (2 mL×2 and 1 mL×6) of 176 NIH-FSH-S1 units of NIADDK-oFSH-17 (Ovagen, ICPbio Reproduction, Auckland, New Zealand) administered twice daily starting 72h before sponge removal. Seven days after oestrus, embryos were recovered by laparotomy, ewes were killed, and uterine horns were processed to study PR expression by immunohistochemistry. The amount of PR was estimated subjectively by 2 independent observers in 5 endometrial compartments: luminal epithelium (LE), superficial (sGE) and deep (dGE) glandular epithelia, and superficial (sS) and deep (dS) stroma. The extent of staining was expressed on a scale from 0 to 100. Data were analysed with a 2×2 factorial ANOVA. Melatonin implants improved fertilization (92v. 57%, for M and C groups, respectively; P<0.01), blastocyst (47v. 9%; P<0.01), viability (88v. 31%; P<0.0001), and freezability (69v. 21%; P<0.001) rates. Specifically, melatonin induced a significant reduction of the number of non-viable (degenerate and retarded) embryos (0.3v. 1.5; P<0.05) and increased blastocysts (2.8v. 0.8; P<0.05) per ewe. Melatonin treatment decreased PR staining intensity (47v. 55%; P<0.05), but this effect was not observed when the individual cell types were compared (Table 1). Because the number of corpora lutea (CL) was responsible for different PR expression in both groups (P<0.0001), animals were divided into 2 ovulation rate categories: <10 CL and ≥10 CL, with lesser PR expression in the ≥10 CL group (P<0.0001); this lower PR immunostaining in ≥10 CL is consistent with progesterone down-regulation of its own receptor. An interaction among number of CL and treatment was found for embryo quality (P<0.05); thus, the positive effect of melatonin on this parameter was particularly effective in the low-ovulation-rate group. These results demonstrate that melatonin treatment in the autumn improves embryo quality in aged ewes, and that this effect is not explained by a differential endometrial sensitivity to progesterone.

Table 1.Embryo production (mean±s.e.m.) in melatonin-treated (M) and control (C) ewes after superovulation in autumn, and staining intensity of progesterone receptors in the endometrium (CL=corpora lutea)
194 Superovulation response does not affect embryo development of pronuclear microinjected embryos in the goat
Authors: Buzzell N. , Blash S. , Miner K. , Schofield M. , Pollock J. , Hawkins N. , Hevy M. , Gavin W.
Published: December 2018

Superovulation of donor animals is essential in the production of transgenic founder goats generated through microinjection. There can be a marked variable response to the exogenous hormones used for superovulation. The objective of this study was to examine how the superovulatory response of individual goats affected the ability of the fertilized, microinjected embryos to develop into offspring. The donors were superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, 2 injections ~12h apart of 32 to 36mg of FSH on Day 12 to 15, progesterone implant removal on Day 14, bred by intact bucks several times starting on Day 15 to 16, an injection of 50μg of gonadotropin-releasing hormone, and surgical collection of 1- to 2-cell embryos from retrograde flushing of the oviduct on Day 17 (~24-48 h, 1-2 days after breeding). Surgical collection allows for an accurate ovulation point (OP) count before the oviduct being retrograde flushed and ova collected and counted. Data from donor animals were grouped by superovulatory response based on OP counts of 1-10, 11-20, 21-30, or >30. The number of donors that contributed per group were 130, 280, 175, and 52, respectively. The recovery rate was 76, 72, 68, and 62%, respectively. After collection, ova were viewed under a dissecting microscope and assessed for fertilization by identifying pronuclei, and 1 pronucleus was microinjected. The fertilization rate was 47, 52, 51, and 56%, respectively. The survivability rate after microinjection was 80, 76, 75, and 76%, respectively. Surviving embryos were transferred (3-5) into recipient goats following a 2- to 6-h in vitro culture (as 1- to 2-cell embryos), allowing for a suitable period to assess viability post-injection. Further in vitro development rates were not assessed because of the short timeframe the embryos stayed in culture. The conception rates were 71, 56, 65, and 53%, respectively, and abortion rates were 23, 10, 14, and 9%, respectively. As some recipients received embryos from multiple donors, this data could not be included in the analysis as identifying which offspring were from the corresponding embryo group could not be confirmed. Data were analysed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). The Wald chi square test under linear regression was used to analyse the number of offspring produced per embryo transferred. No significant differences were found between groups (all P-values were>0.05). This analysis indicated that the range of superovulation response does not affect the developmental competence of the pronuclear microinjected embryo or the ability to produce viable offspring.

Table 1.Comparison of the donor ovulation counts, number of embryos transferred, offspring produced and overall efficiency
195 Effect of feeding a licorice extract to Japanese Black cows on embryo production performance after superovulation treatment
Authors: Aoyagi Y. , Takeuchi M. , Oono Y. , Urakawa M. , Koiwa M.
Published: December 2018

The objective was to determine the effect of feeding a licorice extract that contains glycyrrhizic acid, which is known to have a liver function-enhancing effect, on embryo production performance after superovulation treatment in Japanese Black cows. Japanese Black breed cows (n=136) that had calved 1 to 4 times and had normal uteri as seen by ultrasonography after at least 40 days from the last calving were used as test animals. Animals in the treatment group (n=90) were continuously fed 20g head−1 d−1 of a licorice extract (Kanzou, Fabric Onishi Co. Ltd., Osaka, Japan), with at least 13% glycyrrhizic acid content) once a day, mixed with the formula feed, for 60 to 90 days starting from around the day of calving until ova/embryos were collected. The control group (n=46) received no Kanzou from the day of the last calving until ova/embryo collection. A total of 20 Armour units (AU) of FSH (Antorin R, Kyoritsu Seiyaku Corporation, Kanagawa, Japan) from porcine pituitary was given intramuscularly (IM) twice a day, morning and evening, for 3 consecutive days (decreasing dose schedule: 5 AU×2, 3 AU×2, and 2AU×2) as the superovulation treatment to the cows at 8 to 11 days post-oestrus in both groups. Prostaglandin F2α 25mg and 15mg (IM) were administered in the morning and evening, respectively, on the third day of FSH administration. Artificial insemination was done at 12 and 24h after the start of oestrus, and ova/embryos were collected using a uterine reflux method on Day 7 post-oestrus. The mean numbers of ova/embryos and transferable embryos collected and the mean embryo quality scores (according to the IETS guidelines) in the treatment and control groups were compared by t-test. A chi-squared test was used to compare the proportion of transferable embryos in the 2 groups. There was no significant difference in the mean number of ova/embryos retrieved between the treatment group (21.7±11.8) and the control group (15.8±11.3), although the former tended (P=0.07) to yield more ova/embryos. There was significant difference (P<0.05) in the mean number of transferable embryos collected in the treatment group (13.1±8.3) than in the control group (8.1±5.3). The mean embryo quality score was significantly greater (P<0.01) in the treatment group (1.6±0.4) than in the control group (2.2±0.6). The proportion of transferable embryos in the treatment group (60.3±26.4%) was also significantly higher (P<0.05) than in the control group (51.2±26.0%). In conclusion, feeding a licorice extract to Japanese Black cows for 60 days or longer improves the quality of embryos obtained after superovulation treatment and leads to an increase in the mean number of transferable embryos per cow.
196 Embryo production using follicle-stimulating hormone (FSH) or FSH+equine chorionic gonadotropin in beef donors
Authors: Barajas J. L. , Cedeño A. , Andrada S. , Ortega J. A. , Oviedo J. M. , Tribulo A. , Tribulo R. , Tribulo H. , Bó G. A.
Published: December 2018

The objective of the present study was to evaluate the superovulatory response and embryo production in beef donors using 8 twice-daily injections of FSH or an alternative protocol in which the last 4 FSH injections were replaced with a single injection of eCG. In Exp. 1, 12 mature Bonsmara donor cows, with a body condition score between 3 and 4 (1 to 5 scale) were superstimulated twice every 46 days in a crossover design (i.e. in each experiment all cows received 2 treatments and the 2 treatments were equally represented in each replicate). On Day 0 a.m., all donors received an intravaginal device with 1.2g of progesterone (Diprogest 1200®, Zoovet, Santa Fe, Argentina), along with 50mg of progesterone i.m (Progestar®, Zoetis, Buenos Aires, Argentina) and 5mg of oestradiol-17β (17βOestradiol®, Rio de Janeiro, Argentina) IM. On Day 4 a.m., the superstimulatory treatments were initiated and donors in the control group received 8 applications of FSH (Folltropin-V, Vetoquinol, Lure, France), IM (total dose: 300mg NIH-FSH-P1) in a twice-daily decreasing dosage schedule over 4 days (i.e. 60, 60, 50, 50, 30, 30, 10, and 10mg, respectively). Donors in the FSH+800 eCG group received only the first 4 applications of FSH (i.e. 60, 60, 50 and 50mg, respectively) and on Day 6 a.m. they received 800IU of eCG (Novormón®, Zoetis) IM in a single dose. All donors received 500μg of cloprostenol (Ciclase DL®, Zoetis) IM on Day 6 a.m. and p.m., and the intravaginal devices were removed on Day 7 a.m. All cows also received 100μg of gonadorelin acetate (GnRH, Gonasyn gdr, Zoetis) on Day 8 a.m. and were inseminated with frozen-thawed semen from 2 bulls 12 and 24h later. On Day 15, ova/embryos were collected and evaluated according to the IETS standards. The data were analysed by GLMM (Infostat, 2018). In Exp 2., 18 Bonsmara donors with similar conditions as those in Exp. 1 were superovulated twice in a crossover design. Cows in both groups received similar treatments to those in the FSH+eCG treatment of Exp. 1, except that the total dosage of FSH was 200mg (i.e. 60, 60, 40, and 40mg, respectively) and the eCG given on Day 6 a.m. was either 600IU (group 1) or 800IU (group 2). In Exp 1., the FSH (control) group had a higher (P<0.01) number of fertilized oocytes, but there were no differences in the other end points evaluated (Table 1). In Exp. 2, no differences were found between FSH+800 eCG and FSH+600 eCG groups in any of the parameters evaluated. In conclusion, the replacement of the last 4 injections of FSH by a single dose of eCG decreases the number of treatments required in a superovulation program without negatively affecting the production of transferable embryos.

Table 1.Embryo production (means±s.e.m.) in Bonsmara donors treated with FSH or FSH+eCG
204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes
Authors: Ongaratto F. L. , Rodriguez-Villamil P. , Ganbaatar U. , De Frutos C. , Solin S. , Carlson D. F.
Published: December 2018

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.
205 Towards the correction of meconium ileus with cystic fibrosis transmembrane conductance regulator (CFTR) intestinal expression in CFTR knockout sheep
Authors: Perisse I. Viotti , Fan Z. , Liu Y. , Regouski M. , Van Wettere A. , Wang Z. , Harris A. , White K. L. , Polejaeva I. A.
Published: December 2018

Cystic fibrosis (CF) is a human autosomal genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is responsible for Cl
−
anion transport in epithelial cells. We previously generated CFTR

+/−
and CFTR

−/−
lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. The CFTR

−/−
lambs display many features similar to humans with CF, including meconium ileus (MI), pancreatic fibrosis, portal fibrosis and biliary hyperplasia, small gallbladder, and absence of the vas deferens. Although MI affects only 15 to 20% of human babies with CF, it was observed in 100% of newborn CFTR

−/−
lambs and was the primary cause of death. We here hypothesized that the transgenic expression of ovine CFTR cDNA under regulation of the rat intestinal fatty acid binding protein (iFABP) promoter would promote the correction of MI in CFTR

−/−
sheep. In this study, we constructed the pcDNA3.1>iFABP>CFTR expression vector in order to generate iFABP>CFTR transgenic CFTR

−/−
sheep. PCR and sequence analysis of all junction regions confirmed the presence of the inserts in the vector. We transfected male and female CFTR

−/−
sheep fetal fibroblasts with the construct. Three days after transfection, limiting dilution and G418 selection were used for the isolation of resistant single cell derived colonies. A total of 15 male colonies containing the pcDNA3.1>iFABP>CFTR vector was confirmed by PCR and sequence analysis. Three male cell colonies were used as nuclear donors for SCNT. In total, 129 one-cell-stage cloned embryos were generated and transferred into 11 estrus-synchronized recipients. Four recipients (4/11; 36.3%) were confirmed pregnant at Day 40 to 45 of gestation. One pregnancy went to term and resulted in the birth of a single lamb. The necropsy indicated the same abnormalities, including MI, seen in CFTR

−/−
lambs. A PCR assay confirmed the presence of the iFABP>CFTR transgene in the genome of the cloned offspring. The lack of MI correction is likely due to the low expression of CFTR in this particular colony. Western blot analysis is in progress. Additional embryo transfers will be performed using different single-cell-derived colonies.
22 Vitrification of bovine embryo using antifreeze polyamino acid
Authors: Fujikawa T. , Gen Y. , Hyon S.-H. , Kubota C.
Published: December 2018

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P<0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P<0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P<0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P<0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.