Marcello Rubessa

University of Illinois, Urbana-Champaign | UIUC · Roy J. Carver Biotechnology Center

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 IVF 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 embry...

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...

To compare the efficiency of North Carolina State University medium 23 (NCSU23) and Alpha Minimum Essential Medium (α-MEM) as a base medium, and to evaluate the effects of Anti-Müllerian Hormone (AMH) alone or in combination with Follicle Stimulating Hormone (FSH) on the in vitro development and steroid production of isolated porcine preantral foll...

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 po...

Regenerative medicine approaches for massive craniomaxillofacial 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 n...

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...

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 Dis...

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 b...

Significance The high incidence of human male factor infertility suggests a need for examining new ways of evaluating sperm cells. We present an approach that combines label-free imaging and artificial intelligence to obtain nondestructive markers for reproductive outcomes. Our phase-imaging system reveals nanoscale morphological details from unlab...

The aim of this project was to evaluate the effects of zinc in osteogenic media on differentiation of adipose-derived stem cells (ASC) into osteoblasts. ASC were plated (50,000 cells/mL) and subjected to treatments of zinc at six different concentrations in osteogenic medium: 8 mM, 4 mM, 0.8 mM, 0.4 mM, 0.08 mM, and 0.04 mM, as well as standard ost...

The primary aim of this study was to assess the therapeutic effects of autologous platelet-rich plasma and fibrin scaffolds combined with autologous adipose stem cells (ASCs) for critical-size defects in the pig mandible. Fibrin scaffolds supplemented with calcium hydrogen phosphate and platelet-derived growth factors were hypothesized to accelerat...

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if standard osteogenic medium infused with either vitamin D...

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...

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 spermat...

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 overv...

Cryopreservation of oocytes and embryos is an essential technique for invitro-produced cattle worldwide. One of the great difficulties of cryopreservation of oocytes and blastocysts is the accumulation of lipids in the cytoplasm when produced invitro. The lipid metabolism of oocytes and embryos is classically regulated by the cAMP pathway. Furtherm...

The main objective of an embryo transfer (ET) program is to increase the genetic value of the offspring produced in a given herd. Nutrition, management, and efficiency in the detection of oestrus are among the factors that affect the use of this technology. Protocols that induce oestrus or ovulation allow practitioners to transfer embryos in a pred...

Intrafollicular lipid metabolism is very important for production species such as cattle. Lipids are essential substrates to produce energy during growth, maturation, and acquisition of high competence for the development of oocytes. However, the quantity and distribution of these lipids has been identified as responsible for hindering the process...

The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via co...

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...

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 spe...

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate whether osteogenic medium infused with copper influences th...

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promot...

Bone damage is a common occurrence and although traditional bone healing methods work well, they are not always able to fix all issues pertaining to the injury. However, recent research on stem cell therapies using adipose-derived stem cells (ASC) has shown that ASC that differentiate into osteoblasts can be used as a potentially better solution fo...

Bovine oocyte donor age seems to have an important role on in vitro embryo production. Therefore, the aim of this study was to compare cumulus-oocyte complex (COC) recovery, cleavage, and blastocyst rates between heifers and lactating Holstein donors. A total of 89 animals (heifers: n=60, 11 to 17 months of age; lactating cows: n=29, 60 to 180 days...

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study wa...

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 o...

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 thickn...

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influence...

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-de...

The worldwide production of livestock embryos requires stable medium with long shelf life. In this experiment, we evaluated the impact of the freeze-dried in vitro culture (IVC) medium (Mdry) on in vitro embryo production. We compared the standard IVC and Mdry media for cleavage rate and embryo production. Media solutions (10 mL) were aliquoted int...

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 sub-optimal mechanical competence or bioactivity. We report the use of...

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 tur...

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. Here, we employ spatial light interference microscopy (SLIM) to decouple the thickness and refractive index information of individual cel...

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 mo...

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 A...

The in vitro production of livestock embryos is central to several areas of animal biotechnology. Further, the use of in vitro embryo manipulation is expanding as new applications emerge. ARTs find direct applications in increasing genetic quality of livestock, producing transgenic animals, cloning, artificial insemination, reducing disease transmi...

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 tiss...

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 effici...

In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi...

Reproductive biotechnology frequently requires improvements in our ability to isolate DNA from fewer and fewer cells. It is now possible to find several commercially available kits to meet this demand, but they frequently come at a relatively high cost. The aim of this project was to evaluate the efficiency of a simple handmade protocol to extract...

Sperm selection is a fundamental step for IVF, influencing treatment and the health of offspring. Over the years, several techniques have been tested to recover highly motile sperm, including centrifugation with discontinuous gradients and numerous filtration procedures. In our previous paper, we showed that it is possible, in ~20 min, to select a...

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc...

The in vitro production of bovine embryos has dramatically increased in recent years, and with it the demand of stable media with a long shelf-life. In this experiment we evaluated the impact of the freeze-dried in vitro maturation (IVM) medium (Mdry) on in vitro oocyte maturation. We compared the standard IVM and the Mdry media. Medium M199 was us...

The ability to identify embryos that have the highest developmental potential from a cohort would significantly increase the chances of achieving pregnancy. Metabolic analysis is a well-established analytical approach in biological systems. Starting from this idea, we chose to use high-resolution nuclear magnetic resonance (1H-NMR) spectroscopy. Th...

In Western societies, up to 4% of children born are conceived using assisted reproduction technologies (ARTs), with IVF (in vitro fertilization) now the clinically preferred method of treatment for infertility. It is known that nuclear magnetic resonance (NMR) spectroscopy can be used to identify and quantify metabolite levels in cells and embryos....

The in vitro production of mammalian embryos is central to several areas of biotechnology. Further, the use of in vitro embryo manipulation is expanding as new applications emerge. ART finds direct applications in increasing the genetic quality of livestock, producing transgenic animals, cloning, artificial insemination, reducing disease transmissi...

Conventional label-based contrast enhancement techniques (e.g., fluorescence) frequently modify the genetic makeup of tagged cells, making them poor candidates for use in in-vitro fertilization applications. Instead, we choose a label-free form of contrast, based on interferometric imaging, sensitive to optical path length differences. Compared to,...

The in vitro production of bovine embryos has dramatically increased in recent years. From the first calves produced entirely from IVEP (in vitro embryo production) until today, the in vitro embryo production systems have been modified and improved. The media for embryo production generally are formulated with nutrient excess and furthermore it is...

Objective: Sperm selection is essential to Assisted Reproductive Technology (ART), influencing treatment outcomes and the health of the resulting offspring. Several techniques have been developed to recover a homogeneous population of highly motile sperm cells, including centrifugation gradients, swim up and filtration procedures. It is known that...

Despite the presence of glycogen and triglycerides as energy reserves in bovine embryos, the viability of embryonic cells in culture is maintained primarily by energetic substrates present in the culture medium. The aim of this experiment was evaluate the embryo metabolism during three important stages of development: Days 1 to 3, Days 3 to 5, and...

Bovine leukosis virus (BLV) is a pathogen that affects the bovine immune system and leads to lymphosarcoma, leukemia, decreased milk production, and increased culling rates in cattle. BLV-infected cattle herds can be found worldwide; in the United States, specifically, 38% of beef herds, 84% of all dairy herds, and 100% of large-scale dairy operati...

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected...

The objective of this work was to compare in vitro embryo production of Bos taurus × Bos indicus cross embryos using oocytes from Holstein donors under different production and environment systems. This study also examined the possibility for in vitro production using oocytes imported and transported fresh between the USA and Panama. All animals we...

Recent research has focused on developing secondary, preantral follicles as a new way to collect female gametes to develop in vitro or to study folliculogenesis. Secondary follicles are abundant in ovaries of almost all females, and can be isolated from the ovarian cortex of fresh or cryopreserved tissue. These follicles must be matured to obtain m...

Wide-spread use of IVF has significantly increased the number of multiple births (Janvier et al. 2011 J. Pediatr. 159, 409-413). A potential solution to this problem is to develop improved methods for embryo selection to permit single-embryo transfer. Identification of a noninvasive technique to assess embryo implantation potential in assisted repr...

It has been previously shown that during the pre-implantation phase of embryo development, the pentose phosphate energy pathway is 4 times more active in female embryos when compared with male embryos (Tiffin et al. 1991 J. Reprod. Fertil. 93, 125-132). The different metabolic and growth rates can be attributed to the different expression of X-link...

Reproductive biotechnologies in dromedary camel (Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate...

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157-166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether...

Most systems for producing bovine embryos in vitro use glucose as an energy source despite putative toxic effects. Glucose has a selective embryotoxicity towards female embryos, due to the higher expression of the X-linked glucose-6-phosphate dehydrogenase gene (Kimura et al. 2005 Mol. Reprod. Dev. 72, 201-207). Recently, the replacement of glucose...

Vitrification of in vitro-matured oocytes has important applications in fertility preservation and management of genetic resources. However, despite the increasing interest, the efficiency of oocyte vitrification needs to be improved. It was demonstrated that under stressful conditions of cryopreserving pig oocytes accumulate reactive oxygen specie...

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine...

In vitro-produced embryos are less viable than their in vivo counterparts. It is known that the developmental speed is a reliable marker of embryo viability. One of the major factors impairing in vitro embryo development is oxidative stress. The aim of the study was to evaluate oxidative stress and lipid peroxidation in bovine in vitro-produced emb...

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assi...

The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D)....

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183-1192; Takahashi and First 1992 Theriogenology 37, 963-978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from c...

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335-342; Nandi et a...

The current knowledge on metabolism and glucose utilisation of preimplantation bovine and ovine embryos suggest the reduction of glucose concentration during early culture. On the contrary, it has been demonstrated that glucose is absolutely required for in vitro culture of buffalo embryos, as indicated by the poor efficiency recorded in the absenc...

The aim of this study was to evaluate the effect of exogenous progesterone supplementation on superovulatory response in buffaloes that has undergone a multiple ovulation program. Fourteen Mediterranean buffaloes were divided into two groups and received a 4-day decreasing dosage of an equal mixture of 500 IU of FSH and LH starting on day 8 of the...

The aim of this work was to evaluate chromatin and spindle organization of buffalo and bovine in vitro matured oocytes after vitrification/warming by Cryotop and after their exposure to cryoprotectants (CP). In vitro matured oocytes were vitrified/warmed and exposed to the vitrification/warming solutions containing ethylene glycol (EG), dimethyl su...

The aim of this work was to evaluate the effect of bull on the efficacy of different capacitating agents in buffalo. Spermatozoa derived from 4 different bulls were incubated in absence of a capacitating agent, in presence of 0.01 mM heparin and in 20% buffalo estrous serum (BES) for 2 hours. Sperm were then exposed to lysophosphatidylcholine, an a...