Recent Advances in Biotechnology

The reproductive system of the living organism is the biological system made up of all the anatomical organs involved in sexual reproduction. This system involves the interaction of several fluids and hormones to regulate the functions of the reproductive system. The ultimate goal of the reproductive system is to successfully produce gametes (sperms and oocytes) to attain a combination of genetic material between two individuals, which allows for the possibility of greater genetic fitness of the offspring. In this chapter, we introduce the physiological process of gonadal development, male, and female reproductive system, embryo formation, and development to give the reader the basic concepts for application in the field of assisted reproductive techniques.

Semen analysis has become a definitive tool for understanding the male fertility factor. The volume of the ejaculation indicates the functioning and patency of testicular organs, whereas the quantity and quality of sperm define one’s fertility profile. Thus, semen analysis is the cornerstone for diagnosis as well as for medical management. Semen comes with both cellular and non-cellular components. For the management of male infertility, it is necessary to have better quality sperm. As a result, semen processing has become an integral part of any medically assisted reproduction. There are different standardized preparation methods available that include sperm wash, swim-up technique, pelleting method, density gradient, etc. The aim of this chapter is to discuss semen analysis, its relevance in understanding male fertility factors, and the available sperm preparation methods. Based on the literature, it can be concluded that a proper semen assessment, according to the latest WHO guidelines, is mandatory for diagnosing male fertility issues. The appropriate sperm preparation method and good quality of sperms combined with the skill set of embryologists and environmental conditions will be the decisive factor in any IUI/IVF/ART success.

Sperm freezing is one of the reproductive technologies responsible for increasing domestic animals' fertility and preserving wild animal fertility over the last 100 years. This chapter will shed light on the current understanding of the effect of cryopreservation on sperm cells, advances in cryopreservation in terms of cryoprotectants and freezing protocols for bovines, caprines, ovines, equines, and camelids. Moreover, this chapter will shed light on other technologies needed to be adopted for different animal species.

For conception to occur, whether naturally or via assisted reproductive technology (ART), three things are required; a healthy sperm for fertilization, an ovum released from both ovaries (in the fallopian tubes for natural pregnancy or in the laboratory in IVF/ICSI), and a healthy place for the embryo to implant (a healthy receptive endometrial cavity), so any defect in any of the aforementioned pregnancy requirements will affect the achievement of pregnancy in both types; the natural one or that resulting from ART [1]. Ovarian stimulation is used to overcome any ovulatory disorder or in ART to collect as many eggs as possible (10-15 eggs at least) [2] to maximize the chances of pregnancy. Assessment of ovarian reserve is the first and most important step in ovarian stimulation, as this will indicate the proper method and dose needed for ovulation induction. We test for ovarian reserve using Biomarkers (FSH and AMH) and physical markers (antral follicles count(AFC)) by transvaginal ultrasound(TVS). Both AMH and AFC have similar accuracy in assessing ovarian reserve and are both superior to FSH as they have no cyclic variations [3]. This chapter will discuss all aspects of the physiology of the ovulation process and ovarian stimulation. 

 In the present chapter, the recent studies on artificial insemination (AI) of livestock (large and small) animals via the intrauterine route are summarized. For this, intrauterine inseminations especially in cattle, horse, camel, buffalo, sheep, goat, dog, and cat species will be considered in detail. Brief data on inseminations in other species (pig, turkey, hen, honeybees, silkworm) is also given. Once semen sample is collected from sexually mature and clinically healthy breeding male animals, it has to be kept alive (mainly by dilution and cooling) first and then used either immediately (fresh) or stored (chilled/frozen) until being used in oestrous females. During insertion of the semen (particularly stored ones), it is necessary to place the inseminate in the vicinity of the ovum (Graafian follicle) as close as possible. Hence, fertility results of insemination are always higher when semen is deposited intrauterine. However, this is not as easy as we would expect, due mainly to the anatomical structure of females (cat, dog, sheep, and hen) or viability of semen (fish and camel). To overcome this female- or male-originated limiting factors of fertility, various novel approaches have been reported towards acceptable rates of fertility outcome. Hence, these recent insemination techniques are outlined herein. 

Against all expectations, the presence of a carefully selected normal spermatozoon does not guarantee oocyte activation/fertilization. In contrast, some ICSI cycles will have to face no or low fertilization in several consecutive cycles. Both sperm- and oocyte-derived problems may account for such a dilemma. In case of physiological activation problems, any artificial increase in ooplasmic calcium could rescue the fertilization process. Such approaches are summarized under the term artificial oocyte activation (AOA). AOA can be achieved by modified ICSI techniques, piezoelectrical manipulation, or chemical stimuli. Amongst these approaches, the latter is the currently most accepted one in IVF laboratories around the world and particularly the Ca2+-ionophores ionomycin and calcimycin are the most extensively studied agents. Recently, a ready-to-use ionophore (A23187) has been introduced which is CE-marked and as such will assist in the standardization of AOA techniques. There is growing evidence that for proper indications usage of AOA can be considered quite safe. This conclusion is based on studies on morphokinetics, chromosome segregation, and gene expression. More importantly, available neonatal and neurodevelopmental data are reassuring. However, since artificial oocyte activation rarely results in physiological Ca2+ oscillations and is not beneficial for all patients with a suspected activation deficiency these techniques should not be used without profound indication. 

The embryonic culture system is the main part of in-vitro embryonic development. The culture system consists of chemical factors such as the chemical composition of the culture media and physical factors such as temperature, pH, oil overlay and, incubation systems. The optimal culture conditions are needed to support embryonic development in-vitro, increase pregnancy, implantation rates, and decrease the level of pregnancy loss. Any suboptimality in culture conditions may impair embryonic development and affect subsequent viability. In this chapter, we will focus on the current culture system in the field of assisted reproductive technology (ART), passing by current culture media formulations, deficiencies, culture devices used, and the areas that need further investigations. 

The selected Spermatozoa when it reaches the ovulated Cumulus Oocyte Complex after ovulation, dispersion of the granulosa cells and corona radiata cells occur. The Spermatozoa then must cross the Zona Pellucida (ZP), fuse with the oolemma, and then subsequently fertilize the oocyte. Embryologist Karl Ernst von Baer coined the term ‘Zona Pellucida’ from Greek work Zone which means belt or girdle and Latin work Pellucida which means transparent or shining. This extracellular matrix is about 13-15 um thick and surrounds all the mammalian eggs and pre-implantation embryos. Zona Pellucida structure is made up of carbohydrates, specific proteins, glycoproteins, hyaluronic acid, heparin, collagen, and fibrous proteins. Human Zona Pellucida contains 4 glycosylated proteins namely ZP1, ZP2, ZP3, and ZP4. ZP plays an important role in helping oocytes to transport essential nutrients and helps in avoiding polyspermy by hardening after fertilization. The embryos must break open the protective ZP layer to the implant, the process is called hatching. It is said that in Assisted reproductive treatment (ART) factors such as the non-availability of enzymes from the endometrium which helps in hatching, extended culture, vitrification may lead to failure in the hatching of embryos from ZP. It was postulated that micromanipulation of ZP to create an opening will help the embryos to hatch and thus implant and will lead to an increase in Implantation rates (IR). This process was later called Assisted Hatching (AH). Various methods were discovered for Assisted hatching such as mechanical ZP AH, zona digestion using enzymes, and laser-Assisted hatching. This chapter will focus on the advantages and disadvantages of each method of AH and their applications in ART along with the impact of AH on clinical outcomes. The use of any method of AH should be chosen carefully to avoid damage to the embryo which will defy the whole purpose of application of AH. In any case, laser-assisted hatching is widely used for Pre- Implantation Genetic Testing (PGT) of the embryos as it is very safe if applied properly, convenient, easy to use, and faster compared to other methods of AH. Each laboratory should identify the correct time and stage at which application of AH is considered based on whether it is helping to improve clinical rates or not. 

The availability of cryopreservation technology has extended the scope of human-assisted conception treatment and made it more convenient for patients. Freezing of oocytes and ovarian tissue is now taking place in research institutions and should hopefully become widely available for clinical use in the future if the women have to be away at a critical time during the treatment; the frozen oocyte was thawed and used when required. Good quality embryos frequently remain after the transfer of the required number into the woman; these can be frozen and used at a later date, if required, instead of the couple going through another cycle of in vitro fertilization (IVF) treatment. The conduct of cryopreservation in humans is based on information originally derived from animal work, where sperm cryopreservation, for example, has been carried out for several decades for animal breeding. This chapter will consider the principles of cryopreservation followed by a general description of the practical steps. The clinical applications of cryopreservation will also be described. A final section will deal with frozen embryo replacement.

Hearing the name “Dolly” was and still stirs the minds of professionals and non-specialists towards the term “cloning”, but the way of producing dolly is not the only aspect of cloning. Cloning is defined as the techniques through which identical or virtually identical individuals can be produced. Based on this definition, in this chapter, we are trying to clarify the different applications, aspects, and techniques of cloning such as gene cloning, therapeutic cloning, but to focus on reproductive cloning. Reproductive cloning is the method of making a genetically similar clone of a whole organism. Then it is needed to be discussed with all the scientific thoughts around it, advantages, disadvantages, legal or illegal, and comparing it to other aspects and this is our aim in this chapter.