bluuebunny/crossref_metadata_2025 huggingface dataset:
Research on the variation of resistance of granular system with different sizes under compressive stress
Authors: Zhu Yu , Dong Enyuan , Wang Yongxing , Zhang Liyan , Chen Ru
Published: January 2021

Titanium diboride (TiB2) is a granular material with good electrical conductivity. Its electrical resistance changes significantly under the action of force. Therefore, it can be used as a variable resistor in the power field. In order to achieve this goal, TiB2 particles with a diameter of 30 μm are selected. Through experiments, the resistance of a granular system under a different material height, a different cylinder diameter, and a different force is measured. The relationship between resistance and force is analyzed. Based on the force chain network, a model of the resistance network of the granular system under constant force is established. The effect of cylinder diameter and material height on electrical resistance can be described by this model. The experimental results show the accuracy of the model. The research results of this paper can provide a basis for the application of granular material in the field of power equipment.
Abundance of cavity-free polaritonic states in resonant materials and nanostructures
Authors: Canales Adriana , Baranov Denis G. , Antosiewicz Tomasz J. , Shegai Timur
Published: January 2021

Strong coupling between various kinds of material excitations and optical modes has recently shown potential to modify chemical reaction rates in both excited and ground states. The ground-state modification in chemical reaction rates has usually been reported by coupling a vibrational mode of an organic molecule to the vacuum field of an external optical cavity, such as a planar Fabry–Pérot microcavity made of two metallic mirrors. However, using an external cavity to form polaritonic states might (i) limit the scope of possible applications of such systems and (ii) might be unnecessary. Here, we highlight the possibility of using optical modes sustained by materials themselves to self-couple to their own electronic or vibrational resonances. By tracing the roots of the corresponding dispersion relations in the complex frequency plane, we show that electronic and vibrational polaritons are natural eigenstates of bulk and nanostructured resonant materials that require no external cavity. Several concrete examples such as a slab of the excitonic material and a spherical water droplet in vacuum are shown to reach the regime of such cavity-free self-strong coupling. The abundance of cavity-free polaritons in simple and natural structures points at their relevance and potential practical importance for the emerging field of polaritonic chemistry, exciton transport, and modified material properties.
Molecular dynamics study on flow structure inside a thermal transpiration flow field
Authors: Yamaguchi Hiroki , Kikugawa Gota
Published: January 2021

Thermal transpiration flow is a thermally driven flow from a cold part toward a hot part using a temperature gradient along a wall under a high Knudsen number condition. Many studies have used this type of flow as a pump for microtechnology. The flows adopted in these studies were, in most cases, in the slip or transitional regime. Accordingly, in this research, thermal transpiration flow through a two-dimensional channel with nanoscale clearance in the height direction was studied using the molecular dynamics method. The solid atoms composing the channel walls were explicitly considered. The center part of the nanochannel was controlled as a hot reservoir, whereas both ends of the nanochannel were kept cold. The temperatures of the channel wall atoms were also controlled based on their positions by linearly interpolating the temperature between the hot and cold reservoirs. Two Knudsen number conditions were adopted by changing the width of the computational cell. To study the mean velocity distribution inside the nanochannel, these simulations were performed for 10 ns. We successfully obtained a mean velocity distribution inside the nanochannel, showing the thermal transpiration flow in the vicinity of the channel wall using the pressure-driven counterflow at the center in the height direction even under the dense gas condition. The velocity profile across the nanochannel in the height direction indicated that thermal transpiration flow was induced in the adsorption layer of gas molecules on the channel wall under the dense gas condition.
Nonapproximated third-order exchange induction energy in symmetry-adapted perturbation theory
Authors: Waldrop Jonathan M. , Patkowski Konrad
Published: January 2021

The exchange terms in symmetry-adapted perturbation theory (SAPT) are normally calculated within the so-called S2 or single exchange approximation, which approximates the all-electron antisymmetrizer by interchanges of at most one electron pair between the interacting molecules. This approximation is typically very accurate at the van der Waals minimum separation and at larger intermolecular distances but begins to deteriorate at short range. Nonapproximated expressions for the second-order SAPT exchange corrections have been derived some time ago by Schäffer and Jansen [Mol. Phys. 111, 2570 (2013)]. In this work, we extend Schäffer and Jansen’s formalism to derive and implement a nonapproximated expression for the third-order exchange-induction correction. Numerical tests on several representative noncovalent databases show that the S2 approximation underestimates the exchange-induction contributions in both second and third orders. This underestimation is very similar in relative terms, but the larger absolute values of the third-order exchange-induction effects, and their near complete cancellation with the corresponding induction energies, make the third-order errors more severe. In the worst-case scenario of interactions involving ions, the breakdown of the S2 approximation can result in a qualitatively wrong, attractive character of SAPT total energies at short range {as first observed by Lao and Herbert [J. Phys. Chem. A 116, 3042 (2012)]}. As expected, the inclusion of the full third-order exchange-induction energy in place of its S2-approximated counterpart restores the correct, repulsive short-range behavior of the SAPT potential energy curves computed through the third order.
Polyamorphism in vapor-deposited 2-methyltetrahydrofuran: A broadband dielectric relaxation study
Authors: Gabriel Jan Philipp , Riechers Birte , Thoms Erik , Guiseppi-Elie Anthony , Ediger Mark D. , Richert Ranko
Published: January 2021

Depositing a simple organic molecular glass-former 2-methyltetrahydrofuran (MTHF) onto an interdigitated electrode device via physical vapor deposition gives rise to an unexpected variety of states, as revealed by dielectric spectroscopy. Different preparation parameters, such as deposition temperature, deposition rate, and annealing conditions, lead, on the one hand, to an ultrastable glass and, on the other hand, to a continuum of newfound further states. Deposition below the glass transition temperature of MTHF leads to loss profiles with shape parameters and peak frequencies that differ from those of the known bulk MTHF. These loss spectra also reveal an additional process with Arrhenius-like temperature dependence, which can be more than four decades slower than the main structural relaxation peak. At a given temperature, the time constants of MTHF deposited between 120 K and 127 K span a range of more than three decades and their temperature dependencies change from strong to fragile behavior. This polyamorphism involves at least three distinct states, each persisting for a duration many orders of magnitude above the dielectric relaxation time. These results represent a significant expansion of a previous dielectric study on vapor deposited MTHF [B. Riechers et al., J. Chem. Phys. 150, 214502 (2019)]. Plastic crystal states and the effects of weak hydrogen bonding are discussed as structural features that could explain these unusual states.
A generalized class of strongly stable and dimension-free T-RPMD integrators
Authors: Rosa-Raíces Jorge L. , Sun Jiace , Bou-Rabee Nawaf , Miller Thomas F.
Published: January 2021

Recent work shows that strong stability and dimensionality freedom are essential for robust numerical integration of thermostatted ring-polymer molecular dynamics (T-RPMD) and path-integral molecular dynamics, without which standard integrators exhibit non-ergodicity and other pathologies [R. Korol et al., J. Chem. Phys. 151, 124103 (2019) and R. Korol et al., J. Chem. Phys. 152, 104102 (2020)]. In particular, the BCOCB scheme, obtained via Cayley modification of the standard BAOAB scheme, features a simple reparametrization of the free ring-polymer sub-step that confers strong stability and dimensionality freedom and has been shown to yield excellent numerical accuracy in condensed-phase systems with large time steps. Here, we introduce a broader class of T-RPMD numerical integrators that exhibit strong stability and dimensionality freedom, irrespective of the Ornstein–Uhlenbeck friction schedule. In addition to considering equilibrium accuracy and time step stability as in previous work, we evaluate the integrators on the basis of their rates of convergence to equilibrium and their efficiency at evaluating equilibrium expectation values. Within the generalized class, we find BCOCB to be superior with respect to accuracy and efficiency for various configuration-dependent observables, although other integrators within the generalized class perform better for velocity-dependent quantities. Extensive numerical evidence indicates that the stated performance guarantees hold for the strongly anharmonic case of liquid water. Both analytical and numerical results indicate that BCOCB excels over other known integrators in terms of accuracy, efficiency, and stability with respect to time step for practical applications.
Emerging diseases in international trade in embryos
Authors: Gard Schnuelle Julie
Published: January 2021

A significant change in cattle production and germplasm exchange has occurred over the past 50 years. The growth of artificial reproductive technologies and their broad implementation has become commonplace. The production and subsequent import and export of semen and embryos throughout the world has increased significantly. The embryo transfer industry has reached a new record of growth, with approximately 1.5 million transferrable bovine embryos collected and/or produced in 2018. Over 1 million of these embryos were produced invitro. The increased production of invitro-produced embryos leads to greater opportunities involving international trade. However, further research concerning emerging pathogens is imperative to ensure the efficacy and safety of the embryo transfer industry. Appropriate biosecurity protocols, including reliable testing methodology and effective embryo processing procedures, are key in preventing disease due to emerging and re-emerging pathogens that can be transmitted via embryo transfer.
Heat stress reduces maturation and developmental capacity in bovine oocytes
Authors: Roth Zvi
Published: January 2021

The ovarian pool of follicles, and their enclosed oocytes, is highly sensitive to hyperthermia. Heat-induced changes in small antral follicles can later manifest as impaired follicle development and compromised competence of the enclosed oocytes to undergo maturation, fertilisation and further development into an embryo. This review describes the main changes documented so far that underlie the oocyte damage. The review discusses some cellular and molecular mechanisms by which heat stress compromises oocyte developmental competence, such as impairment of nuclear and cytoplasmic maturation and mitochondrial function, changes in the expression of both nuclear and mitochondrial transcripts and the induction of apoptosis. The review emphasises that although the oocyte is exposed to heat stress, changes are also evident in the developed embryo. Moreover, the effect of heat stress is not limited to the summer; it carries over to the cold autumn, as manifest by impaired steroid production, low oocyte competence and reduced fertility. The spontaneous recovery of oocytes from the end of the summer through the autumn until the beginning of winter suggests that only subpopulations of follicles, rather than the entire ovarian reserve, are damaged upon heat exposure.
Investigation of male and female infertility in llamas and alpacas
Authors: Tibary Ahmed , Campbell Alexis , Rodriguez Jacobo S. , Ruiz Agustin J. , Patino Cristian , Ciccarelli Michela
Published: January 2021

Llamas and alpacas are important production animals in South America, with increasing interest in other parts of the world. Poor reproductive efficiency combined with several unique anatomical and physiological reproductive features offer challenges in the diagnosis and treatment of infertility in camelids. This review presents an approach to the clinical investigation and common causes of infertility and subfertility in the male and female. The selection of males for breeding should be made based on complete evaluation to eliminate congenital and possibly hereditary disorders. Common disorders of the male reproductive system include testicular hypoplasia, testicular and epididymal cysts and testicular degeneration. Semen evaluation presents some challenges owing to the viscous nature of the ejaculate in these species. Females should be screened for congenital genital defects before breeding. Causes of subfertility in the female are dominated by ovarian and uterine disorders. A systematic clinical approach and the use of endometrial biopsy and advanced techniques, such as laparoscopy, allow early identification of these disorders. Further research is needed for continued understanding of the reproductive pathological processes in these species.
Artificially produced gametes in mice, humans and other species
Authors: Hayashi Katsuhiko , Galli Cesare , Diecke Sebastian , Hildebrandt Thomas B.
Published: January 2021

The production of gametes from pluripotent stem cells in culture, also known as invitro gametogenesis, will make an important contribution to reproductive biology and regenerative medicine, both as a unique tool for understanding germ cell development and as an alternative source of gametes for reproduction. Invitro gametogenesis was developed using mouse pluripotent stem cells but is increasingly being applied in other mammalian species, including humans. In principle, the entire process of germ cell development is nearly reconstitutable in culture using mouse pluripotent stem cells, although the fidelity of differentiation processes and the quality of resultant gametes remain to be refined. The methodology in the mouse system is only partially applicable to other species, and thus it must be optimised for each species. In this review, we update the current status of invitro gametogenesis in mice, humans and other animals, and discuss challenges for further development of this technology.
1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development
Authors: Keim J. , Liu Y. , Regouski M. , Stott R. , Singina G. N. , Polejaeva I. A.
Published: January 2021

Invitro maturation is an important process in the invitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and invivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 100UmL−1 penicillin/streptomycin] or maturation medium supplemented with 20ngmL−1 human LIF, 20ngmL−1 IGF1, and 40ngmL−1 FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28M sorbitol fusion medium (0.1mM calcium, 0.5mM magnesium, 0.5mM HEPES, 1gmL−1 bovine serum albumin) by a single pulse of 1.75 kV/cm for 22ms. Reconstructed embryos were activated by exposure to 5μM ionomycin for 5min, followed by 4h of incubation in 2mM 6-(dimethylamino)purine and 10μgmL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean±s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2±2.33%, n=885 vs. 66.8±1.82%, n=822; P<0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2±1.34%, n=259 vs. 90.9±1.22%, n=208; P>0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6±5.1%, n=446 vs. 24.3±2.9%, n=300; P<0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3±20.9%, n=48 vs. 29.0±20.6%, n=31; P<0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined.
This research was supported by UAES project (1343) and RFBR (18-29-07089).
101 Heifer plane of nutrition during the prepubertal phase modifies reproductive development in male fetuses
Authors: O'Callaghan E. , Keogh K. , Staub C. , Cirot M. , Heslin J. , Fair S. , Kelly A. K. , McDonald M. , Kenny D. A. , Lonergan P.
Published: January 2021

Maternal nutritional perturbation during gestation influences fetal development which, in turn, can affect postnatal growth, gonad development, gamete quality, and health of the offspring. The objective of this study was to determine whether maternal diet during the pre-pubertal phase would induce modifications in future male fetal reproductive development. Angus×Holstein-Friesian heifers (n=80) with a mean (±s.d.) age of 141±8d and bodyweight (BW) of 119±23kg had unrestricted access to a perennial ryegrass dominated sward supplemented with either 0.5kg of concentrate daily to achieve an average daily gain (ADG) of 0.50kg (moderate, MOD) or concentrate ad libitum to achieve an ADG of >1.0kg (high, HI). Heifers were offered these divergent dietary regimens from 4.5 to 8.5 months of age, after which they were all offered a moderate plane of nutrition. Heifers were bred to a synchronised oestrus at a mean age of 502±8.2d and mean BW of 360±46.7kg using semen from a single proven sire. Transrectal uterine ultrasonography was carried out 63 days post-AI to determine pregnancy and fetal sex; a cohort of heifers reflective of group average BW and age and carrying male fetuses were slaughtered on Day 100 of gestation (HI: n=10; MOD: n=12). Fetal crown–rump length and weight were recorded. One testis was snap frozen for RNA sequencing and the other was processed for stereology. Mean (±s.d.) fetal crown–rump length and weight were 19.5±0.69cm and 314.6±33.6g, respectively, and did not differ between groups. RNA sequencing revealed 45 differentially expressed genes (DEGs) between groups, 44 of which were upregulated in the HI group. A total of 29 biochemical pathways were identified as significantly enriched (P<0.05), six of which were predicted to be involved in the development of the reproductive tract based on several DEGs. Histological analysis revealed greater (P<0.05) fetal testicular interstitial tissue density in the MOD group and a strong tendency towards increased seminiferous tubule density (P=0.06) in the HI group. In conclusion, offering a high plane of nutrition during the prepubertal period not only increases the rate of sexual development in the heifer but also induces latent in utero modifications to the testicular development of their subsequent male progeny. The consequences for postnatal development of male progeny remain to be elucidated.
This research was supported by Science Foundation Ireland (16/IA/4474).
104 The effect of interspecific hybridization on the development of testes in genus Ovis males
Authors: Volkova L. , Vetokh A. , Volkova N. , Iolchiev B. , Bagirov V. , Zinovieva N.
Published: January 2021

Interspecific hybrids, especially males, are sterile or have reduced fertility. The structure and development of reproductive organs is species-specific. The study of development stages, morphology and biometric parameters in the male testes, the characterisation of species features, and the development of assisted reproductive technologies is of great importance. The aim of this research was to study morphometric parameters of the testes of interspecific hybrids of Argali with sheep of the Romanov breed compared with the original Romanov breed. The objects of research were purebred males of the Romanov breed (n=8) and hybrid males with the following genotypes: 1/4 Argali×3/4 Romanov (Group I, n=10), 1/8 Argali×7/8 Romanov (Group II, n=12). The collection of testes from purebred and hybrid animals was carried out at castration (age=12 months). Testicular tissue was fixed in Bouin’s solution. The following metrics were evaluated: weight and morphometric parameters of the testis, diameter of the seminiferous tubules, and the number of spermatogenic cells in the seminiferous tubule. Statistical analysis (comparison between groups with t-test and ANOVA) was performed using SPSS vs.15.0 (SPPS Inc.). The weight of testes in purebred Romanov males averaged 393±41g. In hybrid animals from groups I and II, this indicator was lower compared with that of purebred animals, by 58% and 41% (P<0.01), respectively. The average circumference of the testes in purebred animals and hybrids of groups I and II was 26±2, 19±3 and 20±3cm. The diameter of the seminiferous tubules in purebred animals and in hybrids of groups I and II was 215±3, 121±2 and 170±6µm, respectively. The number of spermatogenic cells inside the seminiferous tubules affects their size. In purebred animals, this value reached 90±3 cells. Hybrid animals in Groups I and II were inferior to their purebred peers in the number of spermatogenic cells in the seminiferous tubules by 31% (P<0.01) and 9%, respectively. The obtained results indicate the influence of individual characteristics of males on testicular morphology, including the number of spermatogenic cells in the seminiferous tubules.
The studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.
107 Administration of antioxidants improves serum total antioxidant capacity, testosterone, and seminal quality of Magra rams
Authors: Kumar A. , Mehta J. S. , Kumar A. , Purohit G. N.
Published: January 2021

Oxidative stress is one of the most important factors responsible for poor quality of semen, and it is defined as an imbalance between the formation of reactive oxygen species (ROS, including hydroxyl radicals, superoxide anions, and hydrogen peroxide) and antioxidant capacity of semen. Antioxidants act as a powerful defence system against ROS attack in sperm cells by decreasing oxidative stress and they improve semen quality, which could be useful in the management of male infertility. Magra is a dual-purpose (wool and meat) hardy sheep breed that originated in the eastern and southern parts of the Bikaner district of Rajasthan, India. The present study evaluated the effects of the administration of vitamin E with selenium or vitamin C on serum antioxidants, testosterone, and seminal quality of Magra rams. Twenty-one Magra rams, aged between 1.5 and 2.5 years, were randomly assigned to treatment at weekly intervals with administration of either 1mL of vitamin E (tocopherol, 50mg mL−1) and selenium (1.5mg sodium selenite; n=7; VES), 8mL of vitamin C (250mg mL−1 sodium ascorbate; n=7; VC), or 1mL of saline (n=7; control) subcutaneously for 5 consecutive weeks. After the fourth week, semen and blood were collected twice a week for 5 consecutive weeks and assayed for seminal quality parameters, serum testosterone, and serum antioxidants. Data obtained were analysed statistically using te SPSS software (version 25.0; IBM Corp.). Mean values were compared using Duncan’s multiple range test. The semen parameters sperm motility, sperm concentration, live sperm percentage, and% HOST (hypo-osmotic swelling test)-positive sperm were significantly elevated, whereas reaction time and percentage of abnormal sperm were lower in both the treated groups compared with controls (P<0.05). Seminal pH declined significantly in the VES group (P<0.05), whereas it was not different between the VC and control groups of rams (P>0.05). Glutathione peroxidase, superoxide dismutase, and total antioxidant capacity concentrations were higher in serum and seminal plasma of treated rams (P<0.05). Serum testosterone concentrations were significantly elevated in VES and VC groups (P<0.05). We concluded that the administration of vitamin E and selenium or vitamin C improves the serum testosterone production and seminal quality of rams.
108 Synchronization and artificial insemination of South African communal cattle
Authors: Magopa T. L. , Mphaphathi M. L. , Mulaudzi T. , Ramukhithi F. V. , Tshabalala M. M. , Raphalalani Z. C. , Sebopela M. D. , Nkadimeng N. , Sithole S. M. , Nedambale T. L.
Published: January 2021

The present study aimed to evaluate an oestrous synchronization protocol and oestrus response before AI in cows from an organised communal production system. A total of 74 cows Bos indicus (Brahman) and Bos taurus/indicus hybrid (Nguni) type from different communal farmers were screened based on: age (3 to 8 years), body condition score of ≥3 (1–5 scale), not pregnant (excluding heifers), 90 days postpartum, number of parities, good mothering ability, and negative for contagious abortion. Selected cows were subjected to a 9-day OvSynch+CIDR protocol. In brief, the protocol included the insertion of controlled intravaginal drug release (CIDR®, Pfizer Laboratories) devices in the reproductive tract (vagina) containing 1.9g of progesterone in each cow on Day 0 with first oestradiol benzoate (Oestradiol benzoate®, VTech) 2-mL intramuscular (IM) injection. Pregnant mare serum gonadotrophin (PMSG; Chronogest®, Intervet International B.V.) 2.5-mL (IM) injection on Day 5. Removal of CIDR and (IM) injection of prostaglandin F2α (PGF2α; Estrumate®, Intervet South Africa (Pty.) Ltd.) 2mL on Day 8. Following CIDR removal, a heat mount detector (Kamar®) was applied on the individual cow’s tail head for oestrus observation (signs of heat) with second oestradiol benzoate 1-mL (IM) injection on Day 9. AI was performed 36h following withdrawal of the CIDR. Frozen/thawed semen from Bonsmara bulls (n=2) of known and proven fertility was used for AI. The GameteTek Cryo-Mobile laboratory was used during thawing of semen straws. and sperm motility and morphological traits were immediately evaluated by computer-assisted sperm analysis (Sperm Class Analyzer®) before each individual cow was inseminated. Pregnancy diagnosis was performed 90 days after timed AI by ultrasound and transrectal palpation. Data were analysed using the logistic regression procedure of SAS (SAS Institute Inc.), with synchronisation response and conception being treated as binary response variables. All cows were synchronized successfully and an oestrus response rate of 100% was recorded. Pregnancy rates were similar (37.8% vs. 38.9%) for both Bos indicus and Bos taurus/indicus hybrid cattle. Bulls total sperm motility of ≥75% were recorded, following thawing of semen straws. Thus, there was no effect of bull on pregnancy. In conclusion, acceptable oestrus synchronization response was achieved in this communal setup. Superior genetic materials can be successfully introduced through assisted reproductive technologies in organised communal production systems.
113 Different doses of equine chorionic gonadotrophin on pregnancy and twin rates of Bos taurus beef heifers subjected to J-Synch protocol for fixed-time artificial insemination
Authors: Pintos C. G. , Núñez-Olivera R. , Cuadro F. , Brochado C. , Bó G. A. , Menchaca A.
Published: January 2021

The objective was to evaluate the effect of different doses of equine chorionic gonadotrophin (eCG) on pregnancy (P/AI) and twin rates of beef heifers in a progesterone and oestradiol-based treatment (J-Synch protocol) for fixed-time AI (FTAI). A total of 1,404 Bos taurus beef heifers with a body condition score 4.8±0.5 (mean±s.e.m., scale 1–8) were used in three different locations. The presence of a corpus luteum (CL) was determined by ovarian ultrasonography (5.0MHz) at the beginning of the J-Synch protocol. On Day 0, the heifers received 2mg of oestradiol benzoate (intramuscular, IM) associated with an intravaginal device containing 0.5g of progesterone. At device removal, on Day 6, 500µg of cloprostenol sodium was administered IM and the females were randomly assigned to one of two experimental groups to receive 200 (n=702) or 300 (n=702) IU of eCG (Zoetis) IM. Heifers were also tail-painted. On Day 0 females with paint rubbed-off were FTAI 64 h after device removal, and those that remained with the paint received 8 μg of buserelin acetate 64 h after device removal to be FTAI 8 h later. FTAI was performed at 64 to 72h after device removal, and pregnancy diagnosis was performed by ultrasonography 30 to 35 days after insemination. Data were analysed by logistic regression using GLMM. The results are shown in Table 1. Pregnancy rate was not different between heifers treated with 200 or 300IU of eCG (P=0.513) and was not affected by the presence of CL at the beginning of the protocol (P=0.513). Twin rate was greater in heifers treated with 300IU than with 200IU of eCG (P=0.005), and it was not affected by the presence of CL (P=0.362). Interaction between eCG treatment and presence of CL was not significant for pregnancy or twin rates (P=0.532). In conclusion, although 300IU of eCG increased the twin rate compared with 200IU, no differences were found in P/AI. We suggest that the dose of eCG in the J-Synch protocol could be adjusted to 200IU in Bos taurus beef heifers.

Table 1.
Pregnancy per AI (P/AI) and twin rates obtained with different doses of eCG (200 or 300IU) in a J-Synch protocol for fixed-time AI in cycling (CL) and anestrous (no CL) Bos taurus beef heifers

Variable
200 IU
300 IU
P-value

CL
no CL
Total
CL
No CL
Total
eCG
CL
eCG×CL

P/AI (%)
61.6% (135/219)
59.8% (289/483)
60.4% (424/702)
60.2% (133/221)
59.5% (286/481)
59.7% (419/702)
0.513
0.513
0.513

Twin rate (%)
3.0% (4/135)
3.5% (10/289)
3.3% (14/424)
6.8% (9/133)
9.4% (27/286)
8.6% (36/419)
0.005
0.362
0.532
119 Anti-Müllerian hormone as an endocrine marker of fertility in Bos taurus heifers
Authors: Torres-Quijada I. F. , Romo-Garcia S. , Zamorano-Algandar R. , Torres-Simental J. F.
Published: January 2021

The objective of the study was to determine the relationship between the serum concentration of anti-Mullerian hormone (AMH) and fertility indicators in Bos taurus heifers subjected to breeding. From a group of 40 Simmental heifers (15–18 months of age, body condition score of 5 to 7; weight 350–400kg) that received a single dose of 25mg of dinoprost, 23 females entered oestrus and were artificially inseminated 14–16h after onset of the oestrus. If the females returned into oestrus, they were inseminated until conception was confirmed by ultrasonography (30–33d after IA). On Day 4 of the oestrous cycle, a blood sample was collected from each animal, and serum AMH concentration was determined using an ELISA. The AMH levels were classified as normal (AMHn, <0.4ng mL−1) or high (AMHh, >0.4ng mL−1) and were correlated with the number of follicles (NF) and the sampling-to-conception interval (SCi). The number of follicles (antral follicles of 3 to 8mm) in each female was counted at the onset of the recruitment phase during the oestrous cycle. A linear model included AMH level as a fixed effect to analyse NF and SCi. The AMH level was correlated with NF (r=0.8; P<0.001) and SCi (r=−0.61; P<0.05). The AMHh category produced a higher NF (27±3.06 vs. 16±1.85 follicles; P<0.05) and fewer days of SCi (18.22±2.97 vs. 76.30±14.41 days; P=0.03). In conclusion, high AMH levels were related positively with the number of follicles and higher fertility in Bos taurus heifers.
13 Generation of SLICK beef cattle by embryo microinjection: A case report
Authors: Rodriguez-Villamil P. , Ongaratto F. L. , Bostrom J. R. , Larson S. , Sonstegard T.
Published: January 2021

Due to climate change, cattle can experience heat stress more frequently in traditionally temperate, non-tropical environments. Because of this and the fact that most of the world’s inefficient cattle reside in tropical zones, we set out to demonstrate our ability to adapt Angus animals to heat stress through a single gene edit in the prolactin receptor (PRLR). We selected PRLR, because it is known that the SLICK phenotype results from 1 of 3 different mutations in PRLR and therefore is related to heat stress regulation. The overall project was initiated through a partnership between Acceligen and BluePrint Genetics (BPG). BPG harvested 1415 oocytes from a total of 9 ovum pickup rounds from 7 different Angus donors. These oocytes were graded, selected, and put in maturation medium for shipping to Acceligen. Matured oocytes were fertilized in Fert-TALP medium and co-incubated with frozen/thawed semen from 3 different reproductive certified Angus bulls. Consequently, targeted editing was done on a single cell in the zygotes 12h after fertilization by introduction of guide (g)RNA/Cas9 (250ng μL−1 of each) through intracytoplasmic microinjection. Then, treated zygotes (n=1341) were cultured in synthetic oviductal fluid with amino acids (SOFaa) culture medium under a controlled atmosphere until on Day 6, when 277 (20%) grade 1 embryos were selected and 39 returned to Blueprint Genetics for transfer. Because the number of available recipients was limited, only 4 rounds of fresh embryo transfer were completed, and the remaining embryos (n=190) were vitrified for future transfers. Pregnancy checks by ultrasound on Day 30 revealed a 23% (9/39) rate of pregnancy, which decreased to 13% (5/39) by Day 60. In total, 4 animals reached term and delivered healthy calves. Genetic testing of the PRLR target site was done by amplicon sequencing, which showed 3 edited SLICK animals (75%) and one wild-type. Thus, the SLICK zygote editing by microinjection was demonstrated to be an efficient method to produce bovine beef animals and is currently being prepared for regulatory review in multiple countries and commercialization.
140 Effect of addition of different concentrations of L-carnitine during porcine in vitro maturation on embryo quality and development
Authors: Cruzans P. R. , Lorenzo M. S. , Teplitz G. M. , Luchetti C. G. , Lombardo D. M.
Published: January 2021

l-Carnitine (LC) plays an important role in the catabolism of lipids and protects cells from the damage caused by reactive oxygen species due to its antioxidant activity. The aim of this study was to evaluate the effect of adding different concentrations of LC during porcine invitro maturation on embryo quality and development. The cumulus–oocyte complexes were obtained by follicular aspiration from ovaries of slaughtered sows and matured invitro for 44h without LC (control) or with different concentrations of LC (0.6 or 1.25mg mL−1) (Sigma-Aldrich) in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP (cAMP) during the first 22h. Invitro fertilization was performed with fresh boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and cultured in NCSU 23 at 39°C, 7% O2, 5% CO2, and humidity. The cleavage rate was registered on Day 2 and the blastocyst rate on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoescht 33342) and late apoptosis index (TUNEL-positive cells/total cells). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed according to the kit protocol (Roche). LC significantly decreased the cleavage rate (control: 46.2%; LC0.6: 32.1%; LC1.25: 37.9%; P<0.05, Chi-squared test). No significant differences were detected in the blastocyst rate (control: 19.2%; LC0.6: 17%; LC1.25: 10,2%, Chi-squared test) or in number of cells per blastocyst (control: 51.97±3; LC0.6: 56.11±4; LC1.25: 45.62±4, ANOVA). There was embryo hatching in LC treatments but not in the control (control: 0%, LC0.6: 11%; LC1.25: 7.6%). The apoptosis index decreased in LC1.25 compared with LC0.6 (Control: 7,6±1.3%; LC0.6: 10±1.1%; LC1.25: 5,5±0.8%; P<0.05, ANOVA) but there was no significant difference in the apoptosis index between control and LC treatments. In conclusion, LC treatments decreased the cleavage rate but did not modify the blastocyst rate and allowed embryo hatching.
2 Intrafollicular injection of asprosin in water buffaloes and the potential role of FBN1 mRNA and asprosin in follicular function
Authors: Maylem E. R. , Spicer L. , Atabay E. , Atabay E. , Batalha I. , Schutz L.
Published: January 2021

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, whereas the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene; when it is cleaved at the C-terminal end, asprosin is produced. Asprosin acts as an orexigenic hormone and is associated with various metabolic parameters and sex related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells (GC) and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth invivo. In Experiment 1, ovaries were collected from a local slaughterhouse, GC from small (<6mm) and large (6–13mm) follicles were aspirated, RNA was extracted, and gene expression analysis conducted. In Experiment 2, an intrafollicular injection of asprosin (6μL of asprosin in 194μL of phosphate-buffered saline; to achieve 20ng mL−1) or vehicle (200μL of phosphate-buffered saline; Controls) was given via the ovarian stroma below the dominant follicle of synchronized cows (n=5/group) 1 day after injection of prostaglandin F2α, and follicle sizes were measured daily via transrectal ultrasonography until the day of ovulation. Means were compared using t-test for gene expression analysis, and Pearson correlation coefficients calculated among FBN1, OR4M1, and CYP19A1 gene expression. A repeated-measures ANOVA was used to determine the effect of asprosin on follicle size and growth rate of follicles. In Experiment 1, FBN1 mRNA abundance was 7.51-fold greater in GC of small than large follicles (P<0.05). There was no significant difference in the OR4M1 (57.83±39.89 vs. 38.98±4.86) or CYP19A1 (11.46±3.72 vs. 8.27±4.81) mRNA abundance between the 2 sizes of follicles (P>0.10). Abundance of CYP19A1 mRNA was positively correlated with FBN1 (r=0.55, P<0.05) and OR4M1 mRNA (r=0.50, P<0.05). In Experiment 2, there was a treatment×day interaction (P<0.10) for follicle size and growth rate of follicles. Cows treated with asprosin had a higher growth rate from Day 1 to 2 (1.09±0.39 to 2.37±0.32 mm/day) than placebo cows (1.74±0.55 to 1.05±0.61 mm/day) after injection. Most of the follicles from both treatment groups ovulated 3 days post injection. These findings suggest that FBN1 (and thus asprosin) are present in buffalo GC and may be developmentally expressed. Also, asprosin may induce follicular growth when given invivo. Whether these proteins directly regulate aromatase expression, and therefore oestradiol production, during follicle development will require further study.
27 Cryopreservation of bovine semen using cell-permeating antioxidant and protein-free extender
Authors: Yang S. X. , Adams G. P. , Zwiefelhofer E. M. , Rajapaksha K. , Anzar M.
Published: January 2021

Effective semen extenders are those with defined composition that promote sperm longevity. Generation of reactive oxygen species during semen cryopreservation results in sperm membrane lipid peroxidation and reduced longevity. Recent studies demonstrated that small cell permeating dimethyl tyrosine conjugated peptides, such as SS-31, translocate to the mitochondria and scavenge excess reactive oxygen species. The present study was conducted to test the hypothesis that the addition of SS-31 improves the post-thaw quality and fertility potential of bovine semen. The effect of SS-31 was tested in combination with two extenders: (1) conventional tris-egg yolk-glycerol (TEYG, control) and (2) cholesterol-cyclodextrin+tris-glycerol (CC+TG, a defined protein-free extender). Fifteen ejaculates were collected from 5 Black Angus bulls. Ejaculates were diluted to 400×106 spermmL−1 with tris-citric acid buffer, and treated with 0, 50, or 100 µmolmL−1 SS-31 for 15min at 32°C. Semen aliquots were diluted further with TEYG, 0.5mgmL−1 CC+TG, or 1mgmL−1 CC+TG extender to a final concentration of 50×106 spermmL−1. Semen was then cooled to 4°C and frozen in a programmable freezer. Post-thaw sperm motion parameters were evaluated at 0, 2, 4, 6 and 24h with CASA. Semen fertility was determined by fixed-time AI using Hereford-cross cows (n=100). Synchronized cows were inseminated once with semen extended in TEYG, 1mgmL−1 CC+TG, or 1mgmL−1 CC+TG + 100 µmolmL−1 SS-31. Pregnancy was diagnosed by ultrasonography at 27 days post-insemination. Post-thaw sperm motion parameters were compared by ANOVA for repeated-measures, and pregnancy rates were compared using binomial linear mixed-model ANOVA. No differences in sperm motion parameters were detected among SS-31 treatments within extenders. Semen extended in TEYG or 1mgmL−1 CC+TG had greater total and progressive motilities at 0 and 2h post-thaw than semen extended in 0.5mgmL−1 CC+TG (P<0.05). Pregnancy rates after fixed-time insemination did not differ among semen extender groups [TEYG: 15/25 (60%), 1mgmL−1 CC+TG: 19/34 (56%), 100 µmolmL−1 SS-31+1mgmL−1 CC+TG: 21/37 (57%)]. Addition of the cell permeating antioxidant SS-31 did not improve post-thaw semen quality or fertility. The CC+TG extender was as robust as conventional egg yolk extender in protecting bovine sperm during cryopreservation.
This research was supported by NSERC Canada, the Government of Saskatchewan, and Agriculture and Agri-Food Canada.
35 Characterization of the promoter region of ZNFO, an oocyte-specific gene in cattle
Authors: Zhang M. , Baldwin H. , Current J. , Yao J.
Published: January 2021

Maternal factors are essential aspects of oocyte competence, which orchestrate early embryonic development. ZNFO is a Krüppel-associated box domain (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocyte. Previous studies have demonstrated that ZNFO is essential for early embryonic development. However, the mechanisms regulating ZNFO transcription remain elusive. The objective of present study is to elucidate regulatory mechanisms of ZNFO transcription invitro, and specifically to identify putative core promoter and transcription factor(s) regulating ZNFO expression. 5′ Random amplification of cDNA ends (RACE) was performed using RNA isolated from 100 germinal vesicle (GV) stage oocytes to identify the transcription start site (TSS) of ZNFO. To elucidate the molecular mechanisms of ZNFO transcription, a 1.7-kb fragment of the 5′ regulatory region was obtained and cloned into pGL4.14 promoterless vector. The luciferase reporter assay was performed to confirm the promoter activity of the regulatory region. To further identify the core promoter region of the putative ZNFO promoter, a series of 5′ deletions in the ZNFO promoter followed by luciferase reporter assay was performed. The luciferase results indicated that the core promoter region of ZNFO was located within a region 57 to 31bp upstream of the transcription start site. Bioinformatics analysis indicated that a putative USF1/USF2 binding site (GGTCTCGTGACC) is located within the core promoter region. USF1 is a basic helix–loop–helix leucine zipper transcription factor, which regulates the expression of various maternal genes, which are essential for oocyte maturation and early embryonic development in cattle. To confirm that USF1 regulated ZNFO expression, bovine USF1 open reading frame (ORF) was cloned into pcDNA3.1A-HA vector, generating a USF1 overexpression construct. Overexpression of USF1 by transfecting USF1 plasmid enhanced ZNFO promoter activity within HEK293 cells, confirming that ZNFO expression is regulated by USF1. From these results, we conclude that USF1 activates the ZNFO promoter by binding to its target site, GGTCTCGTGACC.
42 Disruption of endogenous SOX2 during porcine embryo development using the CRIPSR/Cas9 system
Authors: Lee M. , Oh J.-N. , Lee D.-K. , Choi K.-H. , Kim S.-H. , Choe G. C. , Jeong J. , Lee C.-K.
Published: January 2021

The lineage specification of the pre-implantation embryo is important to understand the developmental process, but it remains unclear because the expression of lineage-specific genes is distinct among species. Pigs have genetic and physiological traits similar to humans; however, there are differences in gene expression during the pre-implantation stage. To select a candidate gene that affects the formation of the inner cell mass (ICM) in porcine embryo, we conducted preliminary experiments. First, we measured the expression level of candidate genes for lineage specification in parthenogenetic-activated embryos. The expression of pluripotent genes peaked on Day 3 and thereafter decreased gradually. Next, we conducted immunocytochemistry. OCT4 was expressed in all cells in morula and Day 5 blastocyst, but some Day 7 blastocysts expressed OCT4 in both ICM and trophectoderm (TE), whereas others expressed OCT4 only in ICM. NANOG was not observed in the morula stage, whereas SOX2 was located in a restricted area. To examine the effect of SOX2 in ICM formation, we injected plasmid expressing Cas9 and guide (g)RNA using Lipofectamine for efficient transgene expression at the 2-cell stage to increase viability by inducing mosaicism. The expression of enhanced green fluorescent protein (EGFP) contained in the plasmid confirmed that the plasmid was operating normally. In SOX2-knockout (KO) early blastocysts, the numbers of total cells and SOX2- and NANOG-positive cells were greatly decreased, while OCT4 was expressed in most cells. As in early blastocysts, SOX2-KO late blastocysts had fewer cells expressing SOX2, NANOG, and SOX17 than control. To identify the transcriptional consequences of SOX2 reduction, we performed quantitative PCR analysis on non-injected and PX458-gRNA injected blastocysts. Injection of PX458-gRNA resulted in downregulation of NANOG, SOX17, and SMAD7, but not SOX2 and OCT4. Furthermore, proliferation-associated genes were downregulated in injected blastocysts. In conclusion, SOX2-targeted porcine embryos showed blastocoel formation, the inner cell mass formed poorly, and embryos have inefficient cells. Also, the depletion of SOX2 in porcine blastocysts downregulated pluripotent genes and proliferation genes.
This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).