Fertility Cryopreservation

The cryobiology is the science of low temperature biology. Fertility cryopreservation is a vital branch of reproductive science and involves the preservation of gametes (sperm and oocytes), embryos, and reproductive tissues (ovarian and testicular tissues) for use in assisted reproduction techniques (ART). The cryopreservation of reproductive cells is the process of freezing, storage, and thawing of spermatozoa or oocytes. It involves an initial exposure to cryoprotectants, cooling to subzero temperature, storage, thawing, and finally, dilution and removal of the cryoprotectants, when used, with a return to a physiological environment that will allow subsequent development. Proper management of the osmotic pressure to avoid damage due to intracellular ice formation is crucial for successful freezing and thawing procedure. Management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor-recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Cryopreserved cells and tissues can endure storage for centuries with almost no change in functionality or genetic information, making this storage a method highly attractive. There is a pressing need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. There are two major techniques for cryopreservation: freeze-thaw processes and vitrification. The major difference between them is the total avoidance of ice formation in vitrification. However, the biotechnology of the reproduction, although widely implemented, has generated protocols currently used to cryopreserve bovine sperm or oocytes, for example, that are still suboptimal, and cannot readily be extrapolated to other species' gametes. ART provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Nevertheless, it is very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for low survival rates in an effort to develop better cryopreservation. The key factors that affect the life-span of spermatozoa are the combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. On the other hand, oocytes are available only in limited number as compared to spermatozoa, therefore, a cryopreservation protocol must allow a high rate of viability maintenance when they are employed in practical application in ART programs. One of the key factors that influence the freezing process is the ratio of surface area to volume. The oocytes require a longer time to reach osmotic balance with the cryoprotectant solution than the spermatozoa, due to their bigger volume. Then, during cooling of oocytes, various forms of cellular damage may occur, including cytoskeleton disorganization, chromosome and DNA abnormalities, spindle disintegration, plasma membrane disruption and premature cortical granule exocytosis with its related hardening of the zona pellucida. Therefore, animal gametes have been shown to survive storage at low temperatures, and recent results are very encouraging, although reproducible methods have yet to be obtained in many species

Influence of ROS on Ovarian Functions

High level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress (OS) an emerging health risk factor involved in the aging and in many diseases, either in humans or in animals. ROS are a double-edged sword – they serve as key signal molecules in physiological processes, but also have a role in pathological processes involving the female reproductive tract

Reactive Oxygen Species (ROS) and Male Fertility

Oxidative energy production is inevitably associated with the generation of reactive oxygen species (ROS), excessive concentrations of which can lead to cellular pathology. A free radical may be defined as any molecule that has one or more unpaired electrons. The superoxide anion, the hydroxyl radical, and the hypochlorite radical are some of the highest reactive radicals of oxygen. Owing to their high reactivity and to their capability of initiating an uncontrolled cascade of chain reactions, ROS produce extensive protein damage and cytoskeletal modifications and inhibit cellular mechanisms. Aerobic organisms are equipped with a powerful battery of mechanisms that protect them from the adverse effects of lipid peroxidation (LPO) and other manifestations of oxygen toxicity. Defective sperm function frequently causes male infertility, due to abnormal flagella movement, failure to recognize the zona, and inhibition of sperm-oocyte fusion. ROS are fundamental mediators of physiological sperm function, such as signal transduction mechanisms that have an effect on fertility. ROS can have positive effects on sperm and the concentration functions depending on the nature and the concentration of the ROS involved. They are necessary in regulating the hyperactivation and the ability of the spermatozoa to undergo acrosome reaction. An increased amount of superoxide anion (O2-) is one of the first steps required by the spermatozoa for induction and development of hyperactivation and capacitation. Numerous studies have shown that oxidative stress plays an important role in the pathophysiology of infertility and assisted fertility. The paternal genome is of primary importance in the normal embryo and fetal development. ROS-induced sperm damage during sperm translation, such as signal transduction through the seminiferous tubules and epididymis, is one of the most important mechanisms leading to sperm DNA damage. Male germ cells are extremely vulnerable to oxidative stress as the sperm membrane is rich in unsaturated fatty acids and lacks the capacity for DNA repair. Spermatozoa are particularly susceptible to ROS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFA) and their cytoplasm contains low concentrations of the scavenging enzymes. Many clinical and research institutes are investigating the usefulness of antioxidant supplementation and their role in prevention of the infertility problems. Incubation under oxygen in vitro was detrimental to human spermatozoa, decreasing motility and viability. Since then, many reports have associated ROS with impaired sperm function, including decreased motility, abnormal morphology, and decreased sperm-egg penetration. Increasing knowledge of the mechanisms whereby ROS and endogenous antioxidant systems influence reproductive processes can assist to optimize the application of exogenous antioxidants to fertility treatment

Redoxomics and Oxidative Stress: From the Basic Research to the Clinical Practice

Potentially oxidant chemical species, which include not only free radicals but also other oxidizing chemical species such as reactive oxygen species (ROS), for example, hydroxyl radical and hydrogen peroxide, and nitrogen reactive species (RNS), for example, nitric oxide, play a relevant role in all biological processes and especially in cell defenses and molecular signaling. Their action is finely modulated by the antioxidant network that is composed either by endogenous or exogenous compounds (e.g., enzymes, peptides, lipids, and vitamins). An impaired modulation of oxidant species can lead to the so-called oxidative stress that is now considered an emerging health risk factor in almost all living organisms including plants, animals, and humans. Indeed, oxidative stress is related to a reduced lifespan and many diseases (e.g., cardiovascular diseases, neurodegenerative disorders, and metabolic diseases) both in humans and in animals. Unfortunately, oxidative stress does not show any clinical picture, but it can be detected only by means of specific laboratory tests. The recent recognition of a specific “redox code” and the definition of a redoxomics as a new “omics” are now enlarging the horizon of the traditional oxidative stress field leading to the definition of the so-called electrophilic stress. The aim of this chapter is to review the basic principles of redox reaction starting from the concept of free radicals and antioxidant in order to define the “electrophilic stress” as an emerging health risk factor for early aging and almost 1000 illness from infectious diseases to cancer. A paragraph is dedicated to the tests to measure oxidative stress in clinical practice either in humans or in animals in order to prevent, to treat and to monitor electrophilic-related diseases

Assisted Reproductive Technologies in Safeguard of Feline Endangered Species

The growth of the human population and the escalating consumption of natural resources have reduced wild habitats, modifying the existing balance of biological cycles. Therefore, ex situ conservation efforts have received renewed attention as a potential safeguard for species with an uncertain future in the wild. Most wild felid species are classified as rare, vulnerable, or endangered due to poaching and habitat loss. Any directed action taken by humans to enhance animal reproduction results in assisted reproductive technologies (ART) development. These technologies have been included in programs for the conservation of endangered species. Therefore, ART provide a new approach in the safeguard programs of felid biodiversity. Currently, ART mainly include Artificial Insemination (AI); In Vitro Embryo Production (IVEP) consisting of In Vitro Maturation (IVM), In Vitro Fertilization (IVF), In Vitro Culture (IVC), Embryo Transfer (ET), and Intra Cytoplasmic Sperm Injection (ICSI); gamete/embryo cryopreservation; gamete/embryo sexing; gamete/embryo micromanipulation; Somatic Cell Nuclear Transfer (SCNT); and genome resource banking

Ultrasonographic appearance of early embryonic mortality in buffalo (Bubalus bubalis)

Embryonic mortality is one of the main causes responsible of the decline in fertility that occurs in buffaloes during periods of increasing daylight length (out sexual breeding season). Transrectal ultrasonography for pregnancy diagnosis offers some advantages over palpation per rectum: earlier diagnosis of pregnancy/non-pregnancy, determination of embryo/fetus viability, reduction of misdiagnosis, and reduction of .potential. iatrogenic embryo/fetal attrition. Non pregnant buffaloes on Day 25 after AI showed higher Resistive Index (RI) (P<0.05) and Pulsatility Index (P=0.07) values, registered on CL on Days 10 after AI, compared to pregnant buffaloes. RI values were significantly higher (P=0.02) in non pregnant buffaloes also on Day 45 after AI. Colour Doppler sonography could be used to gain specific information relating to the ovarian blood flow in predicting early embryonic loss and to describe the ultrasonographic features of early embryonic death in buffaloes

Effect of Aqueous Extract of Maca Addition to an Extender for Chilled Canine Semen

Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 24 h of refrigerated storage with the higher concentration tested was observed. Although Maca was not able to protect canine semen with extended refrigeration storage time, it increased hyperactivation and preserved DNA integrity in short-term storage

Use of cytobrush for bacteriological and cytological diagnosis of endometritis in mares

Background and Aim: A combined microbial and cytological examination of uterine samples is the main diagnostic method for endometritis in mares. This study aimed to describe a procedure for using the same uterine cytobrush (CB) for both bacteriological and cytological evaluation. Materials and Methods: The procedure consists of rolling the CB onto a sterilized glass slide immediately after collection and before the transfer into a sterile saline solution. In Experiment 1, a comparison between bacteriological results of the cotton swab (CS) and CB or pellet was made in 10 mares; in Experiment 2, bacteriological and cytological results were compared between different processing methods of CB in 28 mares; in other 6 mares, a CB was processed for cytology only, to investigate the reasons for the low cellularity of the pellet. Results: The agreement between culture results from the CB and CS was evaluated, and a comparison between the cytological data obtained by different processing methods of CB was performed. The perfect agreement between the CB and CS microbiological results was found. The described procedure enables useful diagnostic smears for cytology. Moreover, the seeding of both the tip of CB and the saline solution used for the transport produced accurate bacteriological results. Conclusion: The protocol described in this study for the use of CB for both cytological and bacteriological analysis could be used for the diagnosis of endometritis. To maximize diagnostic sample quality, cytology slides must be prepared with meticulous care in the field to preserve cellular integrity and minimize artifacts

Seasonal Variations in the Lipid Profile of the Ovarian Follicle in Italian Mediterranean Buffaloes

The reduced oocyte competence recorded during the non-breading season (NBS) is one of the key factors affecting the profitability of buffalo farming and limits the IVEP efficiency. The purpose of this experiment was to evaluate whether season influences the lipid content within the ovarian follicle in the Italian Mediterranean buffalo. Abattoir-derived ovaries were collected during the breeding season (BS) and the NBS, and different matrices (follicular fluid, oocytes, cumulus and follicular cells) were recovered. After the extraction of the apolar fraction, all samples were analyzed by H1 nuclear magnetic resonance and FF samples by gas chromatography-mass spectrometry. Seasonal differences in lipid composition were observed in all matrices. In particular, during the NBS, the triglyceride content was higher in the follicular fluid and in the oocytes but reduced in the follicular cells. Both cholesterol and phospholipids were reduced in the follicular fluid and follicular cells during the NBS. Furthermore, the total amount of non-esterified fatty acids was significantly increased in the follicular fluid. The seasonal variation in lipid profile of the follicle may, in part, account for the reduced buffalo oocyte competence during the NBS, due to the critical role played by lipids in regulating ovarian functions

Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen.

During cold storage stallion spermatozoa experience undergo oxidative stress, which can impair sperm function and fertilizing capacity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the main endogenous enzymatic antioxidants in stallion seminal plasma, and counteract reactive oxygen species. Semen dilution reduces the endogenous antioxidant concentrations. The aim of this study was to investigate whether addition of 15 IU/mL each of SOD, CAT, and GPX to diluted stallion semen would ameliorate a reactive oxygen-mediated decrease in semen quality during 72 h of storage at 5 °C. Ejaculates (n = 7) were divided in two aliquots and diluted in INRA 96 without (control) or with addition of antioxidants. Semen analysis was performed at the time of dilution and every 24 h during chilled storage. Antioxidant supplementation completely inhibited the storage-dependent increase in activated caspase 3 (P < 0.05). Concomitantly, the antioxidant-supplemented samples had a greater percentage of viable, motile and rapidly moving sperm than control samples after 72 h storage (P < 0.05). The DNA damage, as evaluated by TUNEL assay and SCSA, increased with storage time (P < 0.05). Antioxidant supplementation did not prevent, but did significantly reduce the increase in DNA strand breakage. The results indicate part of the intrinsic apoptotic pathway leading to effector caspase activation was inhibited, although an activation of molecules with endonuclease activity still occurred. In conclusion, adding equal concentrations of SOD, CAT and GPX to a semen extender suppressed caspase-3 activation and improved preservation of stallion sperm motility and viability during 72 h of storage at 5 °C