Label-free, high-throughput holographic imaging to evaluate mammalian gametes and embryos†.

Assisted reproduction is one of the significant tools to treat human infertility. Morphological assessment is the primary method to determine sperm and embryo viability during in vitro fertilization cycles. It has the advantage of being a quick, convenient, and inexpensive means of assessment. However, visual observation is of limited predictive value for early embryo morphology. It has led many to search for other imaging tools to assess the reproductive potential of a given embryo. The limitations of visual assessment apply to both humans and animals. One recent innovation in assisted reproduction technology imaging is interferometric phase microscopy, also known as holographic microscopy. Interferometric phase microscopy/quantitative phase imaging is the next likely progression of analytical microscopes for the assisted reproduction laboratory. The interferometric phase microscopy system analyzes waves produced by the light as it passes through the specimen observed. The microscope collects the light waves produced and uses the algorithm to create a hologram of the specimen. Recently, interferometric phase microscopy has been combined with quantitative phase imaging, which joins phase contrast microscopy with holographic microscopy. These microscopes collect light waves produced and use the algorithm to create a hologram of the specimen. Unlike other systems, interferometric phase microscopy can provide a quantitative digital image, and it can make 2D and 3D images of the samples. This review summarizes some newer and more promising quantitative phase imaging microscopy systems for evaluating gametes and embryos. Studies clearly show that quantitative phase imaging is superior to bright field microscopy-based evaluation methods when evaluating sperm and oocytes prior to IVF and embryos prior to transfer. However, further assessment of these systems for efficacy, reproducibility, cost-effectiveness, and embryo/gamete safety must take place before they are widely adopted.

Rumen-protected methionine supplementation alters lipid profile of preimplantation embryo and endometrial tissue of Holstein cows.

Our objective is to evaluate the effects of feeding rumen-protected Met (RPM) throughout the transition period and early lactation on the lipid profile of the preimplantation embryos and the endometrial tissue of Holstein cows. Treatments consisted of feeding a total mixed ration with top-dressed RPM (Smartamine® M, Adisseo, Alpharetta, GA, United States; MET; n = 11; RPM at a rate of 0.08% of DM: Lys:Met = 2.8:1) or not (CON; n = 9, Lys:Met = 3.5:1). Endometrial biopsies were performed at 15, 30, and 73 days in milk (DIM). Prior to the endometrial biopsy at 73 DIM, preimplantation embryos were harvested via flushing. Endometrial lipid profiles were analyzed using multiple reaction monitoring-profiling and lipid profiles of embryos were acquired using matrix assisted laser desorption/ionization mass spectrometry. Relative intensities levels were used for principal component analysis. Embryos from cows in MET had greater concentration of polyunsaturated lipids than embryos from cows in CON. The endometrial tissue samples from cows in MET had lesser concentrations of unsaturated and monounsaturated lipids at 15 DIM, and greater concentration of saturated, unsaturated (specifically diacylglycerol), and monounsaturated (primarily ceramides) lipids at 30 DIM than the endometrial tissue samples from cows in CON. In conclusion, feeding RPM during the transition period and early lactation altered specific lipid classes and lipid unsaturation level of preimplantation embryos and endometrial tissue.

Repair of critical-size porcine craniofacial bone defects using a collagen-polycaprolactone composite biomaterial.

Regenerative medicine approaches for massive craniomaxillofacial (CMF) bone defects face challenges associated with the scale of missing bone, the need for rapid graft-defect integration, and challenges related to inflammation and infection. Mineralized collagen scaffolds have been shown to promote mesenchymal stem cell osteogenesis due to their porous nature and material properties, but are mechanically weak, limiting surgical practicality. Previously, these scaffolds were combined with 3D-printed polycaprolactone (PCL) mesh to form a scaffold-mesh composite to increase strength and promote bone formation in sub-critical sized porcine ramus defects. Here, we compare the performance of mineralized collagen-PCL composites to the PCL mesh in a critical-sized porcine ramus defect model. While there were no differences in overall healing response between groups, our data demonstrated broadly variable metrics of healing regarding new bone infiltration and fibrous tissue formation. Abscesses were present surrounding some implants and PCL polymer was still present after 9-10 months of implantation. Overall, while there was limited successful healing, with 2 of 22 implants showed substantial levels of bone regeneration, and others demonstrating some form of new bone formation, the results suggest targeted improvements to improve repair of large animal models to more accurately represent CMF bone healing. Notably, strategies to increase osteogenesis throughout the implant, modulate the immune system to support repair, and employ shape-fitting tactics to avoid implant micromotion and resultant fibrosis. Improvements to the mineralized collagen scaffolds involve changes in pore size and shape to increase cell migration and osteogenesis and inclusion or delivery of factors to aid vascular ingrowth and bone regeneration.

Effects of Dietary Supplementation of Conjugated Linoleic Acids and Their Inclusion in Semen Extenders on Bovine Sperm Quality.

Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks -2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates' total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.

"Which Factors Affect Pregnancy Until Calving and Pregnancy Loss in Buffalo Recipients of in vitro Produced Embryos?"

In vitro embryo production and embryo transfer (ET) in buffaloes has been developed for decades. However, most studies are focused on the donor or laboratory improvements, and there is a lack of reports regarding the recipients. Therefore, our aim was to investigate factors associated to pregnancy (P/ET), pregnancy loss (PL), and calving rates in buffalo recipients. The studied factors were season, recipient parity, the synchronization protocol, the CL diameter, asynchrony between the embryo and the recipient, the day of the recipient estrous cycle, the embryo (fresh vs. vitrified), the day of embryo development, and the embryo stage. These retrospective data, from a program of in vitro produced embryos, were analyzed by logistic regression, and the odds ratio was also estimated. Two factors were related to P/ET and the calving rate: (1) progesterone associated to estradiol plus eCG protocol for fixed time ET tended to affect positively P/ET on day 30 (41.9 vs. 36.1%, respectively; P = 0.07; AOR = 1.28) and P/ET on day 60 (37.8 vs. 36.1%, respectively; P = 0.09; AOR = 1.08) compared to the Ovsynch protocol; and (2) the CL diameter (≥14.5 mm) at transfer increased P/ET on day 30 (47.4 vs. 32.5%; P < 0.01; AOR = 1.87) and on day 60 (45.3 vs. 27.7%; P < 0.01; AOR = 2.16), and also the calving rate (37.9 vs. 21.7%; P < 0.01; AOR = 2.20). PL was greater when ET was done in the nonbreeding season compared to the breeding season (PL 30-60: 12.8 vs. 0.0%, P = 0.01; AOR > 999.99; PL 60-calving: 26.8 vs. 3.6%, P = 0.03; AOR = 9.90; and PL 30-calving: 36.2 vs. 3.6%, P = 0.01; AOR = 15.30). In conclusion, the data of our study indicated that the synchronization protocol, the CL diameter, and ET during the breeding season impacted the reproductive efficiency of buffalo recipients.

Emergency ventilator for COVID-19.

The COVID-19 pandemic disrupted the world in 2020 by spreading at unprecedented rates and causing tens of thousands of fatalities within a few months. The number of deaths dramatically increased in regions where the number of patients in need of hospital care exceeded the availability of care. Many COVID-19 patients experience Acute Respiratory Distress Syndrome (ARDS), a condition that can be treated with mechanical ventilation. In response to the need for mechanical ventilators, designed and tested an emergency ventilator (EV) that can control a patient's peak inspiratory pressure (PIP) and breathing rate, while keeping a positive end expiratory pressure (PEEP). This article describes the rapid design, prototyping, and testing of the EV. The development process was enabled by rapid design iterations using additive manufacturing (AM). In the initial design phase, iterations between design, AM, and testing enabled a working prototype within one week. The designs of the 16 different components of the ventilator were locked by additively manufacturing and testing a total of 283 parts having parametrically varied dimensions. In the second stage, AM was used to produce 75 functional prototypes to support engineering evaluation and animal testing. The devices were tested over more than two million cycles. We also developed an electronic monitoring system and with automatic alarm to provide for safe operation, along with training materials and user guides. The final designs are available online under a free license. The designs have been transferred to more than 70 organizations in 15 countries. This project demonstrates the potential for ultra-fast product design, engineering, and testing of medical devices needed for COVID-19 emergency response.

High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa.

The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our knowledge, this is the first reported comparison when using AFM and high-sensitivity interferometric microscopy (such as SLIM) to evaluate spermatozoa. Results indicate that with the use of SLIM microscopy there is similar nanoscale sensitivity as with use of AFM while there is approximately 1,000 times greater throughput with use of SLIM. With SLIM, there is also allowace for the measurement of the dry mass (non-aqueous content) of spermatozoa, which may be a new label-free marker for sperm viability. In the second part of this study, there was analysis of two sperm populations. There were interesting correlations between the different compartments of the sperm and the dry mass in both boars and stallions. Furthermore, there was a correlation between the dry mass of the sperm head and the length and width of the acrosome in both boars and stallions. This correlation is positive in boars while it is negative in stallions.

Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure.

The ability to evaluate sperm at the microscopic level, at high-throughput, would be useful for assisted reproductive technologies (ARTs), as it can allow specific selection of sperm cells for in vitro fertilization (IVF). The tradeoff between intrinsic imaging and external contrast agents is particularly acute in reproductive medicine. The use of fluorescence labels has enabled new cell-sorting strategies and given new insights into developmental biology. Nevertheless, using extrinsic contrast agents is often too invasive for routine clinical operation. Raising questions about cell viability, especially for single-cell selection, clinicians prefer intrinsic contrast in the form of phase-contrast, differential-interference contrast, or Hoffman modulation contrast. While such instruments are nondestructive, the resulting image suffers from a lack of specificity. In this work, we provide a template to circumvent the tradeoff between cell viability and specificity by combining high-sensitivity phase imaging with deep learning. In order to introduce specificity to label-free images, we trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation.

Morphometric analysis of sperm used for IVP by three different separation methods with spatial light interference microscopy.

The goal of this study was to characterize sperm populations resulting from three different methods of sperm selection used for bovine in vitro fertilization. We compared sperm selection with discontinuous Percoll gradients, Swim-Up, and electro-channel. Spatial light interference microscopy (SLIM) was used to evaluate the morphology of the spermatozoa and computer-assisted semen analysis (CASA) was used to evaluate the motility behavior of the sperm. Using these two technologies, we analyzed morphometric parameters and the kinetic (motility) patterns of frozen-thawed Holstein bull spermatozoa after sperm selection. For the first time, we have shown that these methods used to select viable spermatozoa for in vitro fertilization (IVF) result in very different sperm subpopulations. Almost every parameter evaluated resulted in statistical differences between treatment groups. One novel observation was that the dry mass of the sperm head is heavier in spermatozoa selected with the electro-channel than in sperm selected by the other methods. These results show the potential of SLIM microscopy in reproductive biology.Abbreviations: SLIM: spatial light interference microscopy; CASA: computer aided sperm analysis; IVF: in vitro fertilization; BSA: bovine serum albumin; QPI: quantitative phase imaging; IVEP: in vitro embryo production; IACUC: institutional animal care and use committee; CSS: Certified Semen Services; AI: artificial insemination; TALP: Tyrode's Albumin Lactate Pyruvate; MEC: medium for electro-channel; PDMS: polydimethylsiloxane; EC: electro-channel; TM, %: total motility; PM, %: progressive motility; RM, %: percentage of rapid sperm motility; VAP, µm/s: average path velocity; VSL, µm/s: straight-line velocity; VCL, µm/s: curvilinear velocity; ALH, µm: amplitude of lateral head displacement; BCF, Hz: beat cross frequency; STR, %: straightness; LIN, %: and linearity; GLS: generalized least squares; ANOVA: analysis of variance; LSD: Least Significant Difference; SPSS: Statistical Package for the Social Sciences; PCA: principal components analysis.

Label-free microscopy: A non-invasive new tool to assess gametes and embryo quality.

In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species. However, there is no consensus or gold standard method on how to assess their developmental potential. In assisted reproduction the evaluation "problem" is not only limited to embryos but involves the gametes as well. This manuscript provides an overview of some possible applications of label-free microscopy, in particular we describe the potential of the holographic microscopy in the IVF lab. We describe the positive aspects of several currently available microscopy label-free systems. In conclusion, we believe that a next generation of microscopy able to give objective markers for gamete and embryo quality is around the corner.

In Vitro Embryo Production in Buffalo Species (Bubalus bubalis).

In the last years there has been a growing attention in the world regarding buffalo (Bubalus bubalis) breeding. In tropical countries, climatic and environmental conditions make buffalo an irreplaceable milk producer. Since cattle cannot express their own productive potential in these conditions, buffaloes are best suited to satisfy animal protein demand, as they are easy to maintain, long-lived, and parasite resistant. Here we describe how to produce buffalo embryos from slaughterhouse oocytes, with protocols for media formulation, oocyte selection, semen capacitation, and in the end embryo evaluation.

SLIM microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle.

Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analyzed methods to select frozen-thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. To determine the optimal selection method, we used Computer-Assisted Sperm Analysis (CASA). Percoll or Swim-Up were used to select sperm, followed by incubation up to 3 h with the liposome-DNA complexes. The samples were collected after 1 h and after 3 h. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: (1) no incubation, no liposomes and no DNA, (2) incubation with no liposomes and no DNA, (3) incubation with liposomes and no DNA, (4) incubation with liposomes and 1 µg of DNA and (5) incubation with liposomes and 10 µg of DNA. The CASA results for total motility and rapid motility were statistically significant (P < 0.01) between the control and the other treatments in the Percoll group as opposed to Swim-Up. Swim-Up was therefore chosen as the optimal selection method. In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.

Topography and refractometry of sperm cells using spatial light interference microscopy.

Characterization of spermatozoon viability is a common test in treating infertility. Recently, it has been shown that label-free, phase-sensitive imaging can provide a valuable alternative for this type of assay. We employ spatial light interference microscopy (SLIM) to perform high-accuracy single-cell phase imaging and decouple the average thickness and refractive index information for the population. This procedure was enabled by quantitative-phase imaging cells on media of two different refractive indices and using a numerical tool to remove the curvature from the cell tails. This way, we achieved ensemble averaging of topography and refractometry of 100 cells in each of the two groups. The results show that the thickness profile of the cell tail goes down to 150 nm and the refractive index can reach values of 1.6 close to the head.

A Mineralized Collagen-Polycaprolactone Composite Promotes Healing of a Porcine Mandibular Defect.

A tissue engineering approach to address craniofacial defects requires a biomaterial that balances macro-scale mechanical stiffness and strength with the micron-scale features that promote cell expansion and tissue biosynthesis. Such criteria are often in opposition, leading to suboptimal mechanical competence or bioactivity. We report the use of a multiscale composite biomaterial that integrates a polycaprolactone (PCL) reinforcement structure with a mineralized collagen-glycosaminoglycan scaffold to circumvent conventional tradeoffs between mechanics and bioactivity. The composite promotes activation of the canonical bone morphogenetic protein 2 (BMP-2) pathway and subsequent mineralization of adipose-derived stem cells in the absence of supplemental BMP-2 or osteogenic media. We subsequently examined new bone infill in the acellular composite, scaffold alone, or PCL support in 10 mm dia. ramus mandibular defects in Yorkshire pigs. We report an analytical approach to quantify radial, angular, and depth bone infill from micro-computed tomography data. The collagen-PCL composite showed improved overall infill, and significantly increased radial and angular bone infill versus the PCL cage alone. Bone infill was further enhanced in the composite for defects that penetrated the medullary cavity, suggesting recruitment of marrow-derived cells. These results indicate a multiscale mineralized collagen-PCL composite offers strategic advantages for regenerative repair of craniofacial bone defects.

Non-invasive nuclear magnetic resonance analysis of male and female embryo metabolites during in vitro embryo culture.

INTRODUCTION: In the past 20+ years, several studies of bovine embryo production showed how the ratio of male to female embryos changes if embryos are made in vivo or in vitro. It is known that in in vitro systems, the sex ratio is in favor of males when there are high levels of glucose, and favors females when the principal energetic substrate is one other than glucose, like citrate. OBJECTIVES: The aim of this study was to evaluate the embryo metabolism during three important periods of in vitro development: the early development (from day 1 until day 3), the middle of culture (day 3 until day 5), and later development (day 5 until day 7). METHODS: To obtain this information we evaluated the spent medium from each time period by 1H NMR. RESULTS: Our results confirm that embryo metabolism is different between sexes. The new information obtained by identifies markers that we can use to predict the embryo sex. CONCLUSION: These results open a new, non-invasive method to evaluate sex of the embryos before the transfer. In the first period of embryo culture, valine concentration is good indicator (66.7% accurate), while in the last phase of culture, pyruvate depletion is the best marker (64% accurate) to evaluate the sex of the embryo.

Gradient light interference microscopy for 3D imaging of unlabeled specimens.

Multiple scattering limits the contrast in optical imaging of thick specimens. Here, we present gradient light interference microscopy (GLIM) to extract three-dimensional information from both thin and thick unlabeled specimens. GLIM exploits a special case of low-coherence interferometry to extract phase information from the specimen, which in turn can be used to measure cell mass, volume, surface area, and their evolutions in time. Because it combines multiple intensity images that correspond to controlled phase shifts between two interfering waves, gradient light interference microscopy is capable of suppressing the incoherent background due to multiple scattering. GLIM can potentially become a valuable tool for in vitro fertilization, where contrast agents and fluorophores may impact the viability of the embryo. Since GLIM is implemented as an add-on module to an existing inverted microscope, we anticipate that it will be adopted rapidly by the biological community.Challenges in biological imaging include labeling, photobleaching and phototoxicity, as well as light scattering. Here, Nguyen et al. develop a quantitative phase method that uses low-coherence interferometry for label-free 3D imaging in scattering tissue.

Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes.

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the ß-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

High diluted and dynamised follicle stimulating hormone modulates steroid production in isolated porcine preantral follicles cultured in vitro.

OBJECTIVE: This study investigated the effect of two different follicle stimulating hormone (FSH) preparations (diluted/dynamised and diluted) on the in vitro development and steroid production (estradiol, progesterone and testosterone) of isolated porcine preantral follicle after in vitro culture. METHODS: Secondary follicles were cultured in Alpha Minimum Essential Medium (α-MEM+) supplemented with grain ethanol (AL - 0.2%, v/v), diluted/dynamised FSH (rFSH 6cH - 0.05 fg/mL) or diluted-only FSH (1.5 ng/mL) for 4 days. Follicle development was evaluated on the basis of follicular growth, morphology and hormone production. RESULTS: The percentage of follicular integrity and extrusion were not affected by the treatments after culture. For all treatments, follicular diameter increased significantly from Day 0 to Day 4. On Day 2 of culture, the estradiol production was significantly higher in AL and diluted-only FSH treatments compared with diluted/dynamised FSH. However, diluted/dynamised FSH showed a significantly higher progesterone production on Day 2. Only on Day 4, the testosterone production was higher in the AL than diluted-only FSH treatments, but similar to diluted/dynamised FSH treatment. Except for diluted/dynamised FSH treatment, progesterone production increased (P < 0.05) from Day 2 to Day 4; only for AL treatment, a significant increase of testosterone production was observed during culture. CONCLUSION: Compared to control the diluted/dynamised FSH addition increased progesterone production but decreased the estradiol production after in vitro culture of isolated porcine preantral follicles. Taken together the results suggest that at least for progesterone production the mechanism of action of diluted/dynamised FSH differs from its vehicle.

Use of Pig as a Model for Mesenchymal Stem Cell Therapies for Bone Regeneration.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires extreme therapy such as bone grafting or tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice thus far for stem cell therapy for bone regeneration. However, adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC, and both types have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species. The use of scaffolds in combination with stem cells and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Before translation to human medicine, regenerative strategies must be developed in animal models to improve effectiveness and efficiency. The pig presents as a useful model due to similar macro- and microanatomy and favorable logistics of use. This review examines data that provides strong support for the clinical translation of the pig model for bone regeneration.

Modification of the Genome of Domestic Animals.

In the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer. This review looks at the different success rate of these methods and how they have evolved to become more efficient. As well as gene editing technologies, including Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the most recent clustered regulatory interspaced short palindromic repeats (CRISPRs). Through the advancements in gene-editing technologies, generating transgenic animals is now more accessible and affordable. The goals of producing transgenic animals are to 1) increase our understanding of biology and biomedical science; 2) increase our ability to produce more efficient animals; and 3) produce disease resistant animals. ZFNs, TALENs, and CRISPRs combined with gene transfer methods increase the possibility of achieving these goals.