bluuebunny/crossref_metadata_2025 huggingface dataset:
25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes
Authors: Girka E. , Bondioli K. R.
Published: January 2021

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P>0.05). The 2.0μM taxol treatment improved chromosome configuration (P<0.05) with no effect on microtubule distribution compared with the control group (P>0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P<0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate.

Table 1.
Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes

Treatment
n
Normal microtubule distribution (%)
Normal chromosome arrangement (%)

Control
100
44
47

0.5μM Taxol
104
44
37

1.0μM Taxol
98
43
56

2.0μM Taxol
102
49
62a

0.5μM Epothilone B
103
11b
11b

1.0μM Epothilone B
97
6b
8b

2.0μM Epothilone B
100
2b
1b

aP<0.05;. bP<0.001: Different superscripts within a column indicate a significant difference.
33 Effect of exogenous melatonin administration on testicular biometry, libido parameters, seminal parameters, hormone levels, and freezability of Sirohi buck semen during the non-breeding season in Southern Rajasthan
Authors: Sarswat C. S. , Sharma S. , Nehra K. , Sharma P. C. , Parashar M. C. , Vaishnava C. S.
Published: January 2021

The aim of this work was to determine the effect of administration of exogenous melatonin on the testicular biometry, libido parameters, hormone levels, and freezability of semen during the non-breeding seasons in adult Sirohi bucks. Experiments of 4 and 8 weeks were carried out during non-breeding seasons at different times. Twelve bucks, 2 to 3 years of age, were split into melatonin-treated and control groups, with 6 bucks in each group. Throughout the 2 experimental periods, all bucks were permanently kept outdoors under conditions of natural daylength and at ambient temperature. Bucks in the treated group were subjected to single subcutaneous injection of a powder form of melatonin mixed with corn oil at the dose rate 18mg per 50kg on the first day of experiment; bucks in the control group received sterile corn oil injected subcutaneously in control group. Ejaculates were taken from the bucks by artificial vagina (AV) once per week and their reaction time (the elapsed time between exposure to stimuli and first ejaculation) was recorded. Testicular biometry, serum testosterone, and serum melatonin were determined weekly before semen collection. Ejaculates were cryopreserved and thawed weekly and the same quantitative and qualitative variables were measured as for fresh semen samples. Melatonin significantly (P<0.05) improved testicular biometry, libido parameters, and hormone levels compared with the control group during weeks 3 and 4 of the first experiment and from week 5 onward of the second experiment (P<0.05). Semen volume (mL) improved to 0.70±0.039 vs. 0.48±0.009 and 0.94±0.036 vs. 0.56±0.032 in first and second experiments. Greater (P<0.05) progressive motility (%) was noted in the melatonin group (84.59±1.472 and 86.88±0.797) during experiments 1 and 2. Semen quality of each straw from each ejaculate was evaluated after proper thawing within 24h. Mean post-thaw live sperm percentage for experiments 1 and 2 was 69.25±0.382 and 66.90±0.779, progressive motility was 50.42±0.948 and 58.75±1.058, and curled tail sperm count were 51.00±0.715 and 61.36±0.773, respectively. In conclusion, the results of present experiments are novel regarding the use of single subcutaneous melatonin injection during the non-breeding season to improve variables associated with the fertility potential of the Sirohi buck and frozen-thawed seminal parameters.
4 Aryl hydrocarbon receptor targets are upregulated in porcine blastocyst-stage embryos that were cultured invitro: A transcriptional analysis
Authors: Chen P. R. , Spate L. D. , Spollen W. G. , Cecil R. F. , Samuel M. S. , Prather R. S.
Published: January 2021

Invitro-produced (IVP) porcine embryos are developmentally delayed compared with those derived invivo. Thus, efforts have been made to modify the current medium for IVP of porcine embryos in an attempt to shift the abundance of target transcripts towards the invivo level. The objective of the current study was to identify differences in mRNA abundance that may account for reduced developmental competence in IVP embryos and to determine whether alterations to maturation and culture media directed the transcriptional profiles of IVP embryos towards an invivo state. Following AI of gilts, an oviduct and tip of the uterine horn were flushed on Day 2 to recover 4-cell stage embryos; these were cultured for 4 days in MU3, generating IVM and invitro-cultured (IVC) blastocyst-stage embryos. On Day 6, the gilts were killed, and the contralateral horns were flushed to obtain invivo-derived (IVV) blastocyst-stage embryos. The third group of blastocyst-stage embryos, referred to as invitro-matured and cultured (IVMC), were created by aspirating cumulus–oocyte complexes from slaughterhouse-derived ovaries, maturing and fertilizing invitro, and culturing for 6 days in MU3. Total RNA was extracted from pools of 10 blastocyst-stage embryos with 3 replicates per group. First- and second-strand cDNA was synthesised and sequenced by using the Illumina platform (Illumina Inc.). After removal of adapters and reads that mapped to porcine rRNA genes and the PhiX genome, reads were mapped to the Sus scrofa genome by using STAR (version 2.7.1a) with default options. Pairwise comparisons were performed to test for differential expression of genes by using the Bioconductor package DESEqn 2. Transcripts were differentially abundant (false discovery rate <0.05) between IVV and IVC embryos (2,450), between IVV and IVMC embryos (3,045), and between IVC and IVMC embryos (262). Pathways related to cell cycle were downregulated in IVC and IVMC compared with IVV embryos, and pathways related to amino acid transport in metabolism were upregulated in IVC and IVMC compared with IVV embryos. Of particular interest, message for cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was only present in the IVC (956 reads) and IVMC (381 reads) embryos but not in the IVV embryos (0 reads). The aryl hydrocarbon receptor (AHR) promotes transcription of CYP1A1, which encodes a monooxygenase involved in xenobiotic metabolism. Moreover, abundance of 12 other AHR targets was increased in IVC and IVMC embryos (false discovery rate <0.05) compared with IVV embryos. Thus, production of porcine embryos invitro may activate AHR, resulting in altered transcriptional profiles and reduced competence.
This research was funded by USDA-NIFA (2019-67011-29543).
40 Administration of 0.1µM melatonin during invitro maturation of bovine oocytes regulates autophagy levels in produced embryos
Authors: El-Sheikh M. , Mesalam A. A. , Lee K.-L. , Kong I.-K.
Published: January 2021

Melatonin, the antioxidant pineal hormone, is a strong regulator for various cellular processes essential for reproduction. Although the protective role of 0.1µM melatonin against the toxicity of different anti-developmental compounds has been elucidated in numerous studies, its effect on the autophagy level in invitro-produced blastocysts has not been entirely clarified. In this study, oocytes were incubated for 24h in the presence and absence of melatonin, administered during IVM, to investigate the effect of 0.1µM melatonin on the developmental competence of bovine oocytes and pre-implantation embryos, autophagy, and quality of embryos. The developmental potential of embryos were basically the stages from oocytes fertilization to blastocyst production. Gene expression levels were evaluated in matured oocytes, whereas blastocysts were used for immunofluorescence experiments. The differences between treated and control groups were analysed using Student’s t-test (GraphPad Prism version 6; GraphPad Inc.), where P-values <0.05 were considered significant. Results showed that oocyte maturation, Day-4 total cleavage, and Day-8 blastocyst development rates were not significantly improved (melatonin: 72±2 vs. control: 69±2 for cleavage rate, and melatonin: 33±1 vs. control: 31±2 for control for Day-8 blastocyst; P>0.05), whereas the level of reactive oxygen species (ROS) was reduced (P<0.05) with addition of melatonin. Using RT-qPCR, cumulus cells-related (HAS2) and apoptosis-related (Bcl2 and SOD2) genes were upregulated, whereas BAX was downregulated in melatonin-treated oocytes. Using immunofluorescence, apoptosis (caspase-3) and autophagy (Beclin-1 and LC3) markers were underexpressed, whereas the PI3K survival protein (P<0.05) and matrix metalloproteinases (MMP-2 and MMP-9; P>0.05) were overexpressed, in Day-8 embryos of melatonin-treatment. Additionally, the total number of cells per blastocysts, inspected via nuclei-based 4′,6-diamidino-2-phenylindole (DAPI) staining was higher in the melatonin-treated group (P<0.05). Taken together, our study demonstrates that 0.1µM melatonin treatment during IVM does not interfere with developmental competence, but improves the quality of IVF-produced embryos by lowering the incidence of autophagy.
50 Sex ratio at birth in dairy cattle is affected by the invitro embryo production process
Authors: Vivanco-Mackie H. W. , Díaz R. , Ponce-Salazar M. D. , Alayo E. , Bustamante G. , Mesía I.
Published: January 2021

Data from a herd of 3,000 high-producing milking dairy cows under an intensive feeding system was analysed. The IVF laboratory at the farm uses a co-culture system (Vivanco-Mackie; page 30-48 in Proc. 2000 Australian Embryo Transfer Society) and transfers ∼1000 embryos per year. The semen used for IVF and for inseminations (AI) in the herd was female-sexed (F-sexed) and non-sexed (conventional). During the 4 years of evaluation (2016–2019) 1123 calves were born to AI with F-sexed semen and 6,456 to AI with conventional semen; the proportion of males born to AI with F-sexed semen was 9% (91% females), which was not different (P>0.05) from the expected ratio of 10% males and 90% females. The proportion of male calves born to AI with conventional semen was 53.2% (46.8% females), which agrees with the expected ratios for the population. During the same period of evaluation, 805 calves were born to IVF embryos transferred; 240 calves were born from embryos produced using F-sexed semen, of which 29.6% were males and 70.4% females, which was different (P<0.05) from the expected ratio of 10% males and 90% females. The proportion of male calves born from embryo transfer of IVF embryos produced with conventional semen was 82.7%, higher (P<0.05) than the expected 53.2% of males for the population; the proportion of females was 17.3%, significantly lower (P<0.05) than the expected 46.8% females for the population. For IVF, the proportion of males was significantly higher (P<0.05) than expected in all cases; for AI, there was no difference (P>0.05) between the observed sex proportion and the expected values. We conclude that the invitro embryo production process affects the sex ratio at birth in this population, and some factor in the IVF process is affecting the survival to calving of female embryos and consequently increasing the proportion of male calves born from invitro-produced embryos. Gutierrez et al. (2001 Reprod. Fertil. Dev. 13, 361–365) found a similar effect in dairy cattle and linked it to glucose imbalance in the media. Larson et al. (2001 PNAS 98, 9677-9682) found that female cattle embryos had a lower ability to progress in development in glucose-rich media, but the mechanism is not yet fully understood. Xu et al. (1992Mol. Reprod. Dev. 31, 249-252) and others indicate that invitro-produced male bovine embryos predominate among blastocysts, expanded and hatched stages. Meanwhile, embryos lagging in development are predominantly female. Kun Tan et al. (2016 Reproduction 151, 443-543) found that IVF in mice favours the birth of males because of dysregulated expression of representative dimorphic genes. Further studies in cattle are necessary to determine the cause of the increased proportion of males born from invitro embryos and to find ways to control it.
51 Comparison of SexedULTRA 4M and conventional semen for invitro production of bovine embryos using two bulls
Authors: Álvarez-Gallardo H. , Kjelland M. , Pérez-Martínez M. , Villaseñor-González F. , Romo S.
Published: January 2021

SexedULTRA 4M™ (Select Sires) is made using an improved method of sex-sorting sperm in a less damaging environment for retaining sperm integrity through the sorting process. The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this research was to compare conventional (CONV) and SexedULTRA 4M (ULTRA-4M) semen for bovine IVP using 2 bulls. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries from commercial cattle (n=213) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For the IVM, the cumulus–oocyte complexes (COCs) were selected (grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1200, divided equally into 3 replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with both CONV and ULTRA-4M semen from the 2 bulls, separately, at an adjusted concentration of 2×106 and 0.5×106 sperm mL−1, respectively, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2 and 90% N2 at 100% humidity. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. Statistical analysis was carried out with the GLM procedure of SAS (version 9.3; SAS Institute Inc.) to evaluate the results of CONV versus ULTRA-4M for each bull (α level=0.05). Cleavage rates were 57.1%±1.5 and 59.4%±1.5, respectively in CONV and ULTRA-4M groups with Bull 1 and 43.3%±1.5 and 45.2%±1.5 with Bull 2. The percentages of embryos with more than 6 cells were 51.4%±1.0 and 53.9%±1.0, respectively, with CONV and ULTRA-4M with Bull 1 and 30.4%±1.0 and 33.4%±1.0 with Bull 2. The percentage of blastocysts on Day 7 with Bull 1 was 34.4%±1.7 for CONV and 36.2%±1.7 for ULTRA-4M; for Bull 2, the results were 26.5%±1.7 for CONV and 29.5%±1.7 for ULTRA-4M. There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analysed for each bull; however, Bull 1 was significantly superior to Bull 2 for all variables analysed. In conclusion, under the conditions of this research, ULTRA-4M and CONV semen produced similar bovine IVP results.
53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during invitro maturation of oocytes
Authors: Zegarra A. , Rivas J. , Gallegos A. , Mellisho E.
Published: January 2021

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P<0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P>0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.
7 A record of 485 viable cumulus–oocyte complexes recovered and 165 viable embryos produced in a single ovum pickup session from a Senepol breed donor
Authors: Resende A. O. , Bohrer R. C. , Viana J. H. M.
Published: January 2021

The recovery of an exceptionally high number of cumulus–oocyte complexes (COC) in a single ovum pickup (OPU) session is rare, and thus seldom reported in the literature. In the present report, we describe one of these cases. The present data refers to an OPU session performed by author A. O. Resende in a commercial beef farm, located in the rural area of the city of Barretos, Brazil, on October 15, 2019. Breed heifers (N=12) underwent OPU at a random stage of oestrous cycle without any hormonal treatment. Follicle aspirations were performed following the standard operation procedures (SOP) established by ABS Pecplan OPU team, using a portable ultrasound (DP2200, Mindray do Brasil) equipped with a 7.5-MHz microconvex probe, a vacuum pump (WTA), and 18 gauge disposable Jelco catheter (Smith Medical). Invitro embryo production (IVP) was performed at Invitro Technologies laboratory, ABS Pecplan Uberaba, located 140km from the farm. On this day, 1312 oocytes were recovered from the 12 donors. However, a single heifer produced 485 viable COC, from over 500 total COC recovered. The COC from this donor were distributed in four groups, which underwent IVF with Y-sorted semen from four Senepol sires. The invitro embryo production was performed following the SOP established by invitro Technologies, ABS Pecplan. Embryo production was compared among sires by the chi-squared test. Of the 4 sires used during IVF, sire number 4 resulted in a higher blastocyst rate compared with the others (sire 1=25.4%, sire 2=30.6%, sire 3=33.9%, and sire 4=46.3%; P<0.01).The blastocyst rate differed (P<0.01) among sires: 25.4%a (31/122), 30.6%a (37/121), 33.9%a (41/121), and 46.3%b (56/121) for bulls A, B, C, and D, respectively. From the total of 165 viable embryos produced (34.0%), 120 were transferred fresh and the remaining 45 were vitrified using the SOP established by IVB Technologies, ABS. These results highlight the huge potential variation in antral follicle population and number of COC recovered from donors within the same population, breed, and reproductive status. It also shows the potential of IVP in the generation of offspring from high genetic indices.
75 Effect of mycobacterium cell wall fraction immunostimulant on conception rates in heifer recipients
Authors: Palomino J. M. , Cervantes M. P. , Grand F.-X.
Published: January 2021

In cattle, the average pregnancy for IVF embryos around Days 45 to 90 is 35% (Ealy et al. 2019 J. Anim. Sci. 97, 2555–2568). Among other factors, uterine contamination with pathogens from the vagina during embryo transfer may affect pregnancy success. Prophylactic antibiotic treatment for this purpose in embryo recipients is considered malpractice. An alternative to antibiotics is the use of mycobacterium cell wall fraction (MCWF) immunostimulant (Amplimune, NovaVive Inc.) against these pathogens. Therefore, the objective was to determine the effect of MCWF on conception rates in heifer recipients. For this purpose, Holstein heifers were synchronized with 2 injections of prostaglandin F2α 14 days apart and randomly allocated to 2 groups: (1) MCWF (n=292), single intramuscular injection of 5mL of Amplimune on the day of pretransfer oestrus; (2) Control (n=296): no Amplimune treatment. Embryo transfers using quality 1 frozen IVF embryos were performed in all recipients 7 to 8 days after oestrus and with a corpus luteum ≥22mm, using a double sanitary sheath technique. Data were analysed using the generalized linear mixed model (GLMM). Results are shown in Table 1. In conclusion, treatment with MCWF increased the number of recipients to be used for embryo transfer and improved the conception rate at Day 60. Importantly, MCWF treatment was more efficient for getting a recipient pregnant by Day 60 per total of synchronized recipients.

Table 1.
Effect of mycobacterium cell wall fraction (MCWF) on pregnancy in heifer recipients transferred frozen embryos (quality 1)

Variable
MCWF
Control

Utilisation rate
227/292a

174/296b

(No. of transferred recipients/no. of synchronized recipients)
(78%)
(59%)

Pregnancy Day 30
144/227
94/174

(No. of pregnant recipients at Day 30/no. of transferred recipients)
(63%)
(54%)

Pregnancy Day 60
131/227a
83/174b

(No. of pregnant recipients at Day 60/no. of transferred recipients)
(58%)
(48%)

Embryo mortality
13/144
11/94

(No. of embryo loss at Day 60/no. of pregnant recipients at Day 30)
(9%)
(12%)

Pregnancy Day 60 per total of synchronized
131/292a

83/296b

(No. of pregnant recipients/no. of synchronized recipients)
(45%)
(28%)

a,bValues with different superscripts within a row are different (P ≤ 0.05).
81 Pronuclear formation and SMARCA4 incorporation after intracytoplasmic sperm injection (ICSI) or assisted ICSI in pig zygotes
Authors: Briski O. , Gambini A. , Ratner LD , Salamone DF
Published: January 2021

Pigs are considered an important experimental model for their biological similarities to humans, including their potential as organ donors in xenotransplantation. Unfortunately, in this species conventional invitro fertilization results in high polyspermic rates. ICSI avoids polyspermy and ICSI-mediated gene edition could be a powerful technique to produce genetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeller, SMARCA4, translocates to the pronuclei soon after fertilization and its mislocalization or reduction leads to poor embryo development. The aim of this study was to assess whether assisted activation or the use of the piezo drill (PD) during ICSI improves pronuclear (PN) formation rates and to analyse SMARCA4 intensity levels in pronuclei. First, cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured invitro for 44h. Matured and denuded oocytes were subjected to (1) ICSI (n=47), (2) ICSI assisted by PD (ICSIp, n=21), (3) ICSI assisted by electrical activation (ICSIe, n=39), and (4) electrical activation as an haploid parthenogenetic control (HAP, n=21). Presumptive zygotes were fixed for 20min in 4% formaldehyde solution 18h after injection or activation and incubated with SMARCA4 antibody (1:100) and Alexa Fluor (1:1000) as a secondary antibody. Then, the zygotes were classified according to the presence of PN in 2 PN (2-PN), 1 PN with the presence of a semi-condensed or condensed sperm (1-PN), and semi-condensed or condensed sperm with no evidence of PN (no activation). Zygotes that exhibited a different pattern were included in the “other” category. A region of interest was drawn around each PN and the average pixel intensity of SMARCA4 was determined with ImageJ image processing software. Data were analysed by Fisher’s exact test and Kruskal–Wallis test using GraphPad software (GraphPad Inc.). Differences were considered significant at P<0.05. We found no significant differences in 2-PN formation rates among groups after ICSI (ICSI n=16, 34.04%; ICSIe n=10, 25.64%; ICSIp n=6, 28.57%). As expected, the majority of the HAP zygotes exhibited 1 PN (n=14, 66.67%). In contrast, in most of the zygotes of all experimental groups, SMARCA4 was found to be localised in both PN, being absent in polar bodies, metaphase plate, or condensed sperm. Interestingly, out of the total 2-PN porcine ICSI zygotes of all experimental groups (n=25), 7 zygotes (28%) showed clear asymmetric intensity levels between PN. The rest of the ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. In conclusion, our results suggest that neither the use of piezo drill or electrical activation improves PN formation or SMARCA4 pattern. It remains to be determined whether the asymmetric levels of SMARCA4 between PN observed in some zygotes could be associated with a lower embryo developmental competence.
90 Effect of the gonadotrophin-releasing hormone antagonist on the sheep follicular population
Authors: Ferreira H. C. , Vergani G. B. , Bevilaqua J. R. , Rodrigues N. V. , Oliveira M. E. F.
Published: January 2021

The present study was designed to study the follicular population dynamics followed or not by treatment with different doses of the GnRH antagonist in sheep. A total of 18 ewes were submitted to short-term oestrus synchronization protocol (Oliveira et al. 2009 Proc. Braz. Congr. Anim. Reprod.). The animals were 2 or 3 years old, multiparous, and had a body score of 3 to 3.5. On Day 7 after ovulation of synchronized oestrus, females were randomly divided into groups (n=6/group) according to the dose of the gonadotrophin-releasing hormone (GnRH) antagonist (Firmagon®, Ferring Pharmaceuticals) subcutaneously administered: G-control: placebo treatment (administration of saline solution); G-lower dose: 215 µg/kg; and G-higher dose: 235 µg/kg of bodyweight. B-mode ultrasound exams of the ovaries were conducted daily from 1 day before treatment with GnRH antagonist until the females showed oestrous behaviour. Ultrasound equipment (MyLab Vet®, Esaote) was used coupled to a transrectal linear transducer with a frequency of 6 and 8MHz to assess the ovarian population. Data were compared between groups, evaluation days, and their interaction by ANOVA with post hoc using Tukey’s test (P<0.05). There was no interaction (P>0.05) between the studied effects (treatments and evaluation days). The number of small follicles (2–3.49mm) was higher (P=0.0002) in the G-lower dose (5.4±0.4) compared with the G-control (4.1±0.3) and G-higher dose (3.5±0.2). The number of large follicles (≥4.5mm) was lower (P=0.01) in the G-higher dose (0.2±0.0) compared with the G-control (0.5±0.1) and G-lower dose (0.4±0.1). The number of medium follicles (3.5– 4.49mm) and the average diameter of the follicles in the 3 categories of diameter did not differ (P>0.05) between groups. The number of medium follicles differed (P=0.0131) between Days 8 and 15 after synchronized oestrus ovulation. The number of large follicles on Day 6 differed (P=0.0002) of Days 8, 9, 10, 11, 12, 16, and 17. The average diameter of medium follicles differed (P=0.0095) between Days 8 and 10. The number of small follicles and the average diameter of small and large follicles did not differ (P>0.05) between days. In conclusion, the administration of the GnRH antagonist at a higher dose in sheep suppressed the development of large tertiary or antral follicles, whereas at a lower dose, it led to an increase in the population of small follicles.
91 Effect of heat stress during mice germ-cell DNA methylation programming on oocyte developmental competence: A preliminary study
Authors: Moura M. T. , Carvalho C. A. I. , Barros F. R. O. , Mossa F. , Bebbere D. , Paula-Lopes F. F.
Published: January 2021

Heat stress (HS) is characterised by an elevation in body temperature that ultimately undermines organism physiology. Most livestock production occurs in tropical regions under potential HS conditions that diminish productive and reproductive potential. Despite extensive evidence of HS-mediated effects in cell function, stage-specific detrimental effects of HS during oogenesis remain elusive. Mouse models represent an attractive alternative for faster interrogation of stage-specific phenomena during oogenesis. Therefore, the aim of the study was to determine the effects of HS exposure during the major window of female mice germ-cell DNA methylation programming. CD1/Swiss female mice with litters (F0 progeny) at postnatal Day 10 (P10) were randomly allocated to HS (35°C/12-h light; 21°C/12-h dark) or control (CTL: 21°C/24h) for 11 days. The F0 progeny were weaned at P21 and superovulated after reaching puberty at P35. F0 females were superovulated by intraperitoneal injections with 5.0IU of equine chorionic gonadotrophin (PMSG) followed by 5.0IU of human chorionic gonadotrophin (hCG) within a 48-h interval. Pre-implantation embryos were harvested at Day E3.5 in M2 medium under a stereomicroscope. One F0 female per litter was randomly mated to control mice when it reached 6 weeks of age. Data were subjected to least-squares analysis of variance using the General Linear Models procedure of SAS (SAS Institute Inc.). The experiment was replicated twice (CTL: n=4 F0 females and HS: n=4 F0 females). Preliminary results are given as LSM±s.e.m. There was no effect of heat stress on the number of embryos collected per female (CTL: 9.75±4.87 vs. HS: 11.25±4.81) or the percentage of non-viable embryos (CTL: 25.0±0.23% vs. HS: 42.5±0.25%). However, heat stress tended (P=0.07) to reduce the percentage of embryos that reached the morula stage from 63.5±0.08% for CTL to 35.1±0.09% for HS. The percentage of blastocysts collected (CTL: 11.45±0.18% vs. HS: 22.32±0.19%) and litter size of F0 females (CTL: 7.47±1.76 vs. HS: 7.66±1.47) was not affected by treatment. In conclusion, exposure of female mice to 11-day HS during the major wave of de novo DNA methylation during oocyte growth tended to reduce subsequent pre-implantation embryonic development, although it did not affect full-term development after natural mating.
92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos
Authors: Fan Z. , Liu Y. , Perisse I. V. , White K. L. , Polejaeva I. A.
Published: January 2021

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P>0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.
98 Peroxisome proliferator-activated receptor-gamma (PPARG) is dispensable for bovine blastocyst formation
Authors: Quiroga A. C. , de Frutos C. , Zurita E. , Bermejo-Álvarez P.
Published: January 2021

Prostaglandins (PGs) are lipid signalling molecules that play critical roles in gestation by promoting corpus luteus maintenance or luteolysis, and have been suggested to play other roles in early pregnancy, including embryo–maternal crosstalk. The signalling roles of PGs and other lipids are often mediated by peroxisome proliferator-activated receptors (PPARs), transcription factors that regulate the expression of other genes through PPAR-responsive elements. PPARG is a PPAR expressed by bovine pre-implantation embryos whose inhibition by morpholino intrauterine infusion has been reported to impair embryo development. As this approach causes PPARG depletion in both conceptus and uterus, it is unknown whether PPARG-mediated signalling in the embryo is required for embryo development. The objective of this study was to determine whether PPARG is required for blastocyst formation. For that aim, we have evaluated embryo development in PPARG knockout (KO) bovine embryos generated by CRISPR-Cas9 technology. Invitro matured oocytes were allocated in two groups: one was injected with mRNA encoding for Cas9 and sgRNA against PPARG to generate KO embryos (C+G, n=191) and the other was injected with mRNA alone (C, n=148), serving as a microinjection control generating only wild-type embryos. Following fertilization, embryos were allowed to develop to Day 8 blastocysts invitro. No differences were found in cleavage and blastocyst rates between both groups (cleavage 78.5±3.4 vs. 78.4±4.2; Day 7 blastocyst 17.3±3.9 vs. 10.8±2.9; Day 8 blastocyst 20.4±6.2 vs. 16.9±4.7; C+G vs. C; mean±s.e.m.; ANOVA P>0.05). Blastocysts of the C+G group were genotyped by clonal sequencing to determine which embryos in the C+G group were KO (i.e. harboured only frame-disrupting, KO alleles). Twenty-eight out of the 32 blastocysts analysed were edited (87.5%), of which 6 (18.8%) were KO. These results show that PPARG is not required for blastocyst formation, because KO embryos develop to that stage, but do not rule out a possible role in further developmental stages.
99 Increased expression of microRNAs in sperm of Nelore Bulls with high invitro fertility
Authors: Hamilton T. R. S. , Mendes C. M. , Goissis M. D. , Silveira J. C. , Assumpção M. E. O. A.
Published: January 2021

The spermatozoon is no longer known only as a cell that delivers the male genetic material to the oocyte, because it also provides molecules such as microRNAs (miRNAs) that play a significant role in fertilization and embryonic development. MiRNAs are small noncoding RNAs capable of modulating mRNA translation, thus affecting important biological processes. Sperm miRNAs may influence embryo development and therefore, might be related to invitro production of embryos (IVP), considering that individual bulls have different fertility rates when used for IVF. The aim of this work was to identify miRNAs expressed in semen of bulls with high and low IVP rates. The composition of groups was based on a retrospective database from a reproductive biotechnology company between the years of 2016 and 2018, generating around 7000 IVP manipulations of 430 Nelore bulls. We only considered IVP manipulations that used a minimum of 30 oocytes and conventional semen selection by Percoll gradient. A total of 87 Nelore bulls fit these criteria. We then ranked bulls based on cleavage rate (number of cleaved structures/number of oocytes), blastocyst rate (number of blastocysts/number of oocytes) and embryo development rate (number of blastocysts/number of cleaved structures). Considering these three rates, we allocated bulls to two groups. The top eight were considered to have high IVP fertility (HF) and the bottom eight were grouped together as low IVP fertility (LF). We performed the T TEST procedure (SAS 9.3 software; SAS Institute Inc.) to compare the groups for cleavage (P<0.0001), blastocyst (P=0.0006), and embryo development (P=0.0001) rates. For miRNA analysis, sperm were separated using Percoll gradient and subjected to RNA extraction and cDNA synthesis. First, quantitative real-time PCR was used to evaluate the abundance of 380 bovine-specific miRNAs in a pool of samples for each group, using QuantStudio 6Flex. Then, 48 miRNAs presenting at least a 3-fold change of normalized cycle threshold values between groups were selected: 23 highly detected in HF, 1 highly detected in LF, 18 exclusively detected in HF, and 2 exclusively detected in LF. Last, we evaluated the abundance of these selected miRNAs in each experimental unit. We identified four miRNAs highly abundant in sperm from HF bulls, bta-miR-10a, bta-miR-383, bta-miR-93, and bta-miR-449b. Our results suggest that these miRNAs could play important roles in bovine embryo development.
This work was supported by FAPESP (grant# 2018/03871-6).
Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity
Authors: Rayapuram Naganand , Jarad Mai , Alhoraibi Hanna M. , Bigeard Jean , Abulfaraj Aala A. , Völz Ronny , Mariappan Kiruthiga Gayathri , Almeida-Trapp Marilia , Schlöffel Maria , Lastrucci Emmanuelle , Bonhomme Ludovic , Gust Andrea A. , Mithöfer Axel , Arold Stefan T. , Pflieger Delphine , Hirt Heribert
Published: January 2021

Significance

Mitogen-activated protein kinases (MAPKs) function in all eukaryotes in signaling extracellular stimuli to intracellular responses and ultimately link them to chromatin events by targeting transcription factors and chromatin remodeling complexes. In plants, MAPKs play crucial roles in immunity, development, and stress responses, but so far no attempts have been made to identify phosphorylation of chromatin-associated proteins. By using a phosphoproteomic approach on MAPK mutants, we identified a number of chromatin-associated MAPK substrates and characterize an AT-hook motif containing nuclear localized (AHL) DNA-binding protein 13 in plant immunity and demonstrate that phosphorylation regulates AHL13 protein stability and, in turn, its function in response to pathogens.
Forensic Human Image Identification Using Medical Indicators
Authors: Zeng Jinhua , Qiu Xiulian , Shi Shaopei , Bian Xinwei
Published: January 2021

Diseases not only bring troubles to people’s body functions and mind but also influence the appearances and behaviours of human beings. Similarly, we can analyse the diseases from people’s appearances and behaviours and use the personal medical history for human identification. In this article, medical indicators presented in abnormal changes of human appearances and behaviours caused by physiological or psychological diseases were introduced, and were applied in the field of forensic identification of human images, which we called medical forensic identification of human images (mFIHI). The proposed method analysed the people’s medical signs by studying the appearance and behaviour characteristics depicted in images or videos, and made a comparative examination between the medical indicators of the questioned human images and the corresponding signs or medical history of suspects. Through a conformity and difference analysis on medical indicators and their indicated diseases, it would provide an important information for human identification from images or videos. A case study was carried out to demonstrate and verify the feasibility of the proposed method of mFIHI, and our results showed that it would be important contents and angles for forensic expert manual examination in forensic human image identification.
Effects of rebinding rate and asymmetry in unbinding rate on cargo transport by multiple kinesin motors
Authors: Wang Yao , Liu Yu-Ying , Liang Jian , Wang Peng-Ye , Xie Ping
Published: January 2021

Many intracellular transports are performed by multiple molecular motors in a cooperative manner. Here, we use stochastic simulations to study the cooperative transport by multiple kinesin motors, focusing mainly on effects of the form of unbinding rate versus force and the rebinding rate of single motors on the cooperative transport. We consider two forms of the unbinding rate. One is the symmetric form with respect to the force direction, which is obtained according to Kramers theory. The other is the asymmetric form, which is obtained from the prior studies for the single kinesin motor. With the asymmetric form the simulated results of both velocity and run length of the cooperative transport by two identical motors and those by a kinesin-1 motor and a kinesin-2 motor are in quantitative agreement with the available experimental data, whereas with the symmetric form the simulated results are inconsistent with the experimental data. For the cooperative transport by a faster motor and a much slower motor, the asymmetric form can give both larger velocity and longer run length than the symmetric form, giving an explanation for why kinesin adopts the asymmetric form of the unbinding rate rather than the symmetric form. For the cooperative transport by two identical motors, while the velocity is nearly independent of the rebinding rate, the run length increases linearly with the rebinding rate. For the cooperative transport by two different motors, the increase of the rebinding rate of one motor also enhances the run length of the cooperative transport. The dynamics of transport by N (N = 3, 4, 5, 6, 7 and 8) motors is also studied.
Damping-like effects in Heisenberg spin chain caused by the site-dependent bilinear interaction
Authors: Zhang Yu-Juan , Zhao Dun , Li Zai-Dong
Published: January 2021

We investigate a continuous Heisenberg spin chain equation which models the local magnetization in ferromagnet with time- and site-dependent inhomogeneous bilinear interaction and time-dependent spin-transfer torque. By establishing the gauge equivalence between the spin chain equation and an integrable generalized nonlinear Schrödinger equation, we present explicitly a novel nonautonomous magnetic soliton solution for the spin chain equation. The results display how the dynamics of the magnetic soliton can be controlled by the bilinear interaction and spin-polarized current. Especially, we find that the site-dependent bilinear interaction may break some conserved quantity, and give rise to damping-like effect in the spin evolution.
Interplay of parallel electric field and trapped electrons in kappa-Maxwellian auroral plasma for EMEC instability
Authors: Nazeer M , Qureshi M N S , Shen C
Published: January 2021

In this paper, propagation characteristics of electromagnetic electron cyclotron (EMEC) waves based on kappa-Maxwellian distribution have been investigated to invoke the interplay of the electric field parallel to the Earth’s magnetic field and auroral trapped electrons. The dispersion relation for EMEC waves in kappa-Maxwellian distributed plasma has been derived using the contribution of the parallel electric field and trapped electron speed. Numerical results show that the presence of the electric field has a stimulating effect on growth rate, which is more pronounced at low values of wave number. It is also observed that as the threshold value of trapped electron speed is surpassed, it dominates the effect of the parallel electric field and EMEC instability is enhanced significantly. The electric field acts as another source of free energy, and growth can be obtained even in the absence of trapped electron drift speed and for very small values of temperature anisotropy. Thus the present study reveals the interplay of the parallel electric field and trapped electron speed on the excitation of EMEC waves in the auroral region.
Asymmetric multiple-image cryptosystem based on LUP decomposition in spherical diffraction domain
Authors: Zhang Ziyi , Wang Jun , Wu Hanmeng , Zhu Zheng , Chen Xu-Dong , Hu Yuhen
Published: January 2021

In this paper, an asymmetric multiple-image cryptosystem is proposed using LUP decomposition (LUPD) in the spherical diffraction (SpD) domain. To the best of our knowledge, the SpD transform is applied to the optical encryption for the first time. In the proposed cryptosystem, multiple grayscale images are encoded into a sparse and binary amplitude distribution with the cascaded LUPD and SpD transform. The matching private keys are generated by LUPD, which is convenient for administrative permissions by setting different numbers of private keys. Since LUPD and SpD are both non-linear processes, high security can be achieved to resist attacks such as the retrieval attack. In addition, up to 12 plaintext images can be encrypted with high decryption quality using the proposed cryptosystem. On the other side, the sparse and binary ciphertext can be stored or transmitted with 1/8 space compared with general 8-bit-grayscale ciphertext, which is a merit of using LUPD. The feasibility and effectiveness of the proposed asymmetric cryptosystem are verified by numerical simulation results.