The cryobiology of spermatozoa.

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above-agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics.

Advances in porcine assisted reproductive technology (ART) make it possible to use cryopreserved sperm, embryos and somatic cells in the maintenance, relocation and regeneration of swine genetics. In this review, development of key application-limiting technology is discussed in each cell type, focusing on the efficiencies, ease of storage and transportation, and minimization of pathogen transmission. Methods to regenerate swine genetics and/or models using frozen sperm, embryos and somatic cells in combination with other porcine ARTs, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transplantation (SCNT), are also discussed. The applications of these ARTs utilizing cryopreserved cells will greatly increase the efficiency as well as biosecurity for maintenance, relocation and rederivation of swine genetics/models.

Regulation of oocyte mitochondrial DNA copy number by follicular fluid, EGF, and neuregulin 1 during in vitro maturation affects embryo development in pigs.

Little is known about mitochondrial DNA (mtDNA) replication during oocyte maturation and its regulation by extracellular factors. The present study determined the effects of supplementation of maturation medium with porcine follicular fluid (pFF; 0, 10%, 20%, and 30%) on mtDNA copy number and oocyte maturation in experiment 1; the effects on epidermal growth factor (EGF; 10 ng/mL), neuregulin 1 (NRG1; 20 ng/mL), and NRG1 + insulin-like growth factor 1 (IGF1; 100 ng/mL + NRG1 20 ng/mL), on mtDNA copy number, oocyte maturation, and embryo development after parthenogenic activation in experiment 2; and effects on embryo development after in vitro fertilization in experiment 3. Overall, mtDNA copy number increased from germinal vesicle (GV) to metaphase II (MII) stage oocytes after in vitro maturation (GV: 167 634.6 ± 20 740.4 vs. MII: 275 131.9 ± 9 758.4 in experiment 1; P < 0.05; GV: 185 004.7 ± 20 089.3 vs. MII: 239 392.8 ± 10 345.3 in experiment 2; P < 0.05; Least Squares Means ± SEM). Supplementation of IVM medium with pFF inhibited mtDNA replication (266 789.9 ± 11 790.4 vs. 318 510.1 ± 20 377.4; P < 0.05) and oocyte meiotic maturation (67.3 ± 0.7% vs. 73.2 ± 1.2%, for the pFF supplemented and zero pFF control, respectively; P < 0.01). Compared with the control, addition of growth factors enhanced oocyte maturation. Furthermore, supplementation of NRG1 stimulated mitochondrial replication, increased mtDNA copies in MII oocytes than in GV oocytes, and increased percentage of blastocysts in both parthenogenetic and in vitro fertilized embryos. In this study, mitochondrial biogenesis in oocytes was stimulated during in vitro maturation. Oocyte mtDNA copy number was associated with developmental competence. Supplementation of maturation medium with NRG1 increased mtDNA copy number, and thus provides a means to improve oocyte quality and developmental competence in pigs.

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system.

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Vascular endothelium-specific overexpression of human catalase in cloned pigs.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction.

During the last few decades in vitro production of mammalian embryos and assisted reproductive technologies such as embryo transfer, cryopreservation, and cloning have been used to produce and propagate genetically superior livestock. However, efficiencies of these technologies remain low. For these technologies to become more commercially viable, the efficiencies must improve. Despite this importance of reproduction for the livestock industry, little progress in decreasing embryonic mortality has been made. The livestock industry has succeeded in achieving large increases in average milk production of dairy cattle, growth rate in beef cattle and leanness in swine but reproductive efficiency has actually decreased. For example, research has provided little progress toward developing an objective method to examine viability of a single living embryo. At the same time, the growth of miniaturization technologies beyond integrated circuits and toward small mechanical systems has created opportunities for fresh examination of a wide range of existing problems. While the investigation and application of miniaturization technologies to medicine and biology is progressing rapidly, there has been limited exploration of microfabricated systems in the area of embryo production. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review the progress in demonstrating microfluidic systems for in vitro embryo production (IVP) and embryo manipulation. Microfluidic technology could have a dramatic impact on the development of new techniques as well as on our basic understanding of gamete and embryo physiology.

Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts.

The soybean phytoestrogen genistein has a range of estrogenic actions demonstrated in various species; however, only limited research has been done to investigate its effects in swine. The objective of this study was to characterize the effects of a graded dose of genistein on estrogen-sensitive uterine and cervical tissues in ovariectomized gilts. Thirty-four postpubertal gilts were ovariectomized and assigned randomly to 1 of 6 treatment groups 15 d postovariectomy. Treatment groups received vehicle, estradiol benzoate (2 mg/d), or genistein (50, 100, 200, or 400 mg/d) via intramuscular injection at 12-h intervals for 10 d. Following the treatment period, gilts were euthanized, and uterine and cervical tissues were collected and processed for chemical or histological analysis. Uterine and cervical tissue mass, as indicated by wet, dry, and protein weights and total DNA content (expressed per 100 kg of BW), increased as the dosage of genistein increased (P < 0.001 for each regression). Uterine and cervical wet weights were increased by a dosage of 200 mg of genistein/d (P < 0.001 and P < 0.01, respectively) but not by 100 mg of genistein/d (P = 0.38 and P = 0.14, respectively) compared with those of control gilts. Height of epithelial cells lining the uterine glands and the lumen of uterus and cervix increased when gilts were treated with estradiol benzoate or 400 mg of genistein/d (P < 0.01). When the gilts were treated with estradiol benzoate or 400 mg of genistein/d, immunohistochemical staining demonstrated an increase in the percentage of cells that stained positive for progesterone receptor in the uterine glands and in the cells lining the vaginal cervix (P < 0.05). In gilts treated with 400 mg of genistein/d, the percentage of cells stained positive for proliferating cell nuclear antigen increased in the epithelium of the uterine glands, uterine lumen, and vaginal cervix (P < 0.05). Tissue growth was stimulated by genistein in a dosage-dependent manner, although no dosage of genistein induced a response as great as that of estradiol benzoate. Estrogen-sensitive tissues of the ovariectomized gilt, such as the cervix and uterus, are affected by injection of large dosages of the phytoestrogen genistein. The sensitivity of the uterus of the gilt to estrogenic substances makes it a potential model to examine the impact of environmental endocrine modulators on reproductive tissues.

Transgenic animals in biomedicine and agriculture: outlook for the future.

Transgenic animals are produced by introduction of 'foreign' deoxyribonucleic acid (DNA) into preimplantation embryos. The foreign DNA is inserted into the genetic material and may be expressed in tissues of the resulting individual. This technique is of great importance to many aspects of biomedical science including gene regulation, the immune system, cancer research, developmental biology, biomedicine, manufacturing and agriculture. The production of transgenic animals is one of a number of new and developing technologies that will have a profound impact on the genetic improvement of livestock. The rate at which these technologies are incorporated into production schemes will determine the speed at which we will be able to achieve our goal of more efficiently producing livestock, which meets consumer and market demand.

Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro.

The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.

Transgenic technology and applications in swine.

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce "new" genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or "cloning" strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.