Advances in Frontier Reproduction and Endocrinology in Animal

With a long history from demonstration that fertilizing power resides in the spermatozoa and not in the liquid portion of semen by Lazanno Spalbanzani, 1780, the phenomenal growth of AI occurred in the 1940s in the United States. The procedures developed in the United States became established worldwide for manual placement of semen in the reproductive tract of the female by a method other than natural mating. Concept of AI has given rise to a group of technologies commonly known as “Assisted Reproduction Technologies” (ART), whereby offspring are generated by facilitating the meeting of gametes (spermatozoa and oocytes) which could contribute to overcome specific causes of infertility in animals and to produced offspring of desired genetic trait. ART may also involve the transfer of the products of conception to a female, for instance if fertilization has taken place in vitro or in another female (in vivo). Other recent techniques encompassed by ART include the following:

Artificial insemination: Artificial insemination is the procedure involving the mechanical deposition of pre-collected semen into the uterus of an estrous female by a technician. The widespread commercial application of AI, a robust tool in livestock breeding, began in earnest in the 1950’s after researchers developed methods to successfully freeze cattle semen. This technology, which is the most extensively applied reproductive biotechnology in the world, has many valuable advantages such as reducing venereal diseases, rapidly increasing genetic merits of the production animals through selective breeding and eliminating lethal alleles. The potential to pass high genetic merits of a selected male to thousands of females makes this a far more efficient technology for producing large numbers (1.5 x 10⁸) of offspring per year compared to female based technologies, such as embryo transfer (ET), which can only produce a few progeny from a selected female. More recently, the advent of separation of sperm into X- and Y-chromosome fractions using flow cytometry has added a new dimension to livestock production.

Since the first report of live rabbit pups of predetermined sex using X and Y-enriched sorted semen over 20 years ago, this sperm sexing technology has proven to be a reliable and more efficient method of obtaining predetermined sex in animals compared to other relatively wasteful methods of embryo sexing and pre-implantation genetic diagnosis. Live offspring born from fresh sex- sorted semen have been reported in many species including cattle, pigs, horses and sheep. As the sorting procedure can separate sperm only at a speed of approximately 10 million per hour, inseminations have to be carried out using limited numbers of sperm in each insemination dose compared to the conventional standards for AI. To obtain high success rates with sorted semen AI, most trials have been carried out using fresh sorted semen, nevertheless, use of frozen sorted semen is ultimately more beneficial to the livestock industry as it can be stored for long duration and easy to transport anywhere in the world. 

Semen sexing is the process of separating spermatozoa into two subpopulations containing X-chromosome and Y-chromosome bearing spermatozoa. In theory, use of the X-bearing spermatozoa fraction for insemination will result in female progeny, while the Y-bearing spermatozoa fraction will give male progeny. Considerable efforts have been made in this area over the past 30-40 years, but only recently has there been considerable progress, particularly in cattle. The most commonly applied technique for the separation of a fresh semen sample into X and Y-chromosome bearing fractions is fluorescence activated cell sorting (FACS). The concept behind this technique is described below:

Fluorescence activated cell sorting (FACS): This technique involves the separation of X- and Y- bearing spermatozoa in small quantities based on the DNA content of the spermatozoa so that they can then be used in in vitro fertilization and AI programs. The procedure is relatively slow and requires expensive infrastructure at least for mass scale commercial application. The technique is based on the biological principle that the DNA content of the X and Y chromosomes as X-chromosome has 3.8% more DNA than Y-chromosome. This difference can, nonetheless, be detected with FACS technique. Before the spermatozoa are passed through the cell sorter, the sample is exposed to a nontoxic DNA dye that specifically binds to DNA to allow measurement of the DNA content of each spermatozoa. The cell suspension is then passed through an extremely fine nozzle that vibrates at high frequency causing the fluid stream to disperse into micro-drops which, optimally holds only one spermatozoa after the conditions are calibrated. When the stream of micro-droplets is passing through a UV laser beam, the already stained DNA will start to glow or emit fluorescence. The strength (intensity) of the florescence is measured by a detector, and if the florescence is in the range designated for the Y-bearing spermatozoa, that micro-drop is given an electric charge. Furthermore, if the intensity is within the range for X-bearing spermatozoa, the micro-drop is given an opposite electric charge. The charged micro-drops fall between two charged plates, resulting in two groups of droplets to be separated into different pools (Figure). Droplets that fall outside of the fluorescence range for X or Y are collected into a third group where the sex cannot be determined. The disadvantages of the technique are its relatively slow process and increased wastage of unsorted spermatozoa. The advantage is it can give up to 95% purity sexed semen samples. 

Figure: Schematic representation of the concept behind the Florescent Activated Cell Sorting technique used in separation of X- and Y-bearing chromosomes in a fresh semen sample. (Adopted from Alexander B, Mastromonaco G, King WA (2010) Recent Advances in Reproductive Biotechnologies in Sheep and Goat. J Veterinar Sci Technol 1:101. doi:10.4172/2157-7579.1000101) 

It is a variation of IVEP where a single sperm directly into the ooplasm of a matured oocyte using a microscopic needle. In natural mating, genetically important but biologically inferior (e.g., low sperm production, poor sperm motility and sperm abnormalities) males cannot be used. ICSI besides providing an understanding of the chronology consequences during sperm and oocyte activation during the first cell cycle of embryo development (Galli et al. 2003) also ICSI would be an effective technique to use on genetically important but biologically inferior male gametes for procreating domestic and wild livestock species (Keskintepe et al., 1997) and thereby facilitating the production of large number of embryos and offspring from a single genetically valuable animal asooplasmic microinjection bypasses all natural barriers to sperm penetration and removes all biological selectivity from the process, such as sperm binding to the Zona Pellucida and fusion of the sperm with the oolemma (Gordon and Laufer, 1988) which impair the fertilizing ability of a genetically important but biologically inferior male.

In livestock industry, the ICSI technique could be an alternative approach to generate genetically modified organism, using sperm as the DNA carrier (Perry et al., 1999, 2001), for propagation of useful genetic trait (Wang et al., 2003) or predetermination of sex (Hamano et al., 1999). The progeny produced by this technique could be used as founder animals for the production of recombinant protein in their milk such as pharmaceutical proteins for the treatment or prevention of human diseases or biomaterials for medical use (Niemann and Kues, 2003; Keefer, 2004). Therefore, engaging in ICSI appears to be profitable and beneficial to livestock industry and till now, ICSI technique is one of the modern ARTs which has been successfully used in humans, rodents and other animals, especially in the cattle (Nasar and Rahman, 2010). 

In vitro embryo production (IVEP) has been identified as a reproductive technology with the potential to produce more offspring from genetically valuable animals than standard Multiple Ovulation and Embryo Transfer (MOET) (Baldassarre and Karatzas, 2004; Cognie et al., 2004).At birth, each ovary contains hundreds of thousands of oocytes; most are lost through atresia, which starts even before birth. This tremendous loss of genetic material from genetically important animals could be reduced by harvesting oocytes from the ovary and using IVEP techniques (Brackett and Zuelke, 1993; Hasler, 1998). Besides preventing the loss of superior genetic trait this technique can further be used to provide an excellent source of embryos for emerging scientific research (Gordon and Lu, 1990) like embryo sexing, cloning, nuclear transfer, transgenesis etc, further it also provide a tool for study developmental potential of embryos including the pattern of gene expression and cytogenetic disorders during the development (Galli and Lazzari, 2008) and efforts to produce embryonic stem cells.

In IVEP technique oocytes were removed from the donor animals (live or death) ovaries and the oocytes mature in an incubator and are fertilized with sperm. The resulting zygotes incubate and develop in the laboratory before being placed into the recipient cow. Recent application of ultra sound guided transvaginal oocyte retrieval (TVOR) and oocyte pick up (OPU) has providing a better scope for IVEP and provide maximum utilization of donors with superior genetic traits by repeated collection oocytes from live donors in order to propagate such genetic traits (Callensen et al., 1987).

The initial purpose of commercial IVEP was to obtain viable embryos from females that may not be able to produce progeny through conventional techniques. However, at present IVEP could be applied to exploit the production potentiality of   females that will not respond to superovulatory treatments, fail to produce transferable embryos for ET programme, or possess abnormalities in their reproductive tracts (i.e. ovarian adhesions or blocked fallopian tubes), for females that are fatal(age, accident, disease, etc.), or that are pregnant heifers and cows during the first trimester of gestation, and for heifers and cows with and without calf during the first one, two or three months after calving (post-partum period) and also has applications for normal cyclic heifers and cows, and prepuberal calves to reduce the generation interval. Recent use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex (Wheeleret al., 2006). 

The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers' sex selection dreams reality in the 1990s and now semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter (Wheeler et al., 2006). Flow cytometric sorting instrumentation to sort X- and Y-bearing sperm resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation (Johnson, 2000).Predetermination of the sex of offspring could have a significant impact on livestock production, particularly in selection programmes where the products (e.g. milk) comes from only one female animal and it has also advantages in the situation where to establish a herd or flock of specified genotype, as when an exotic breed or species is to be introduced.

Several methods have been used to reach this objective which is presented in Table4.The result and accuracy of most of these techniques are satisfactory, and according to the established objective, it is convenient to opt for a pre-selection (sexing semen or embryo) as opposed to post-selection (foetus) methods of sex. In the case of sexing embryos, the only method used routinely on a commercial scale is to biopsy embryos and amplify Y-chromosome -specific DNA using polymerase chain reaction. This method is effective for more than 90%of embryos and is > 95% accurate (Seidel Jr. 1999). 

Table 4. Different methods of sexing.

It is the most recent evolution of selective assisted breeding in animal husbandry. Cloning animals is a reliable way of reproducing superior livestock genetics and ensuring herds are maintained at the highest quality possible.  It’s important to remember that cloning does not manipulate the animal’s genetic makeup nor change an animal’s DNA. It is simply another form of assisted reproduction. Cloning allows livestock breeders to create an exact genetic copy of an existing animal, essentially an identical twin. Clones are superior breeding animals used to produce healthier offspring.

A new era in reproductive biotechnology began when Wilmut and colleagues produced the first somatic cell nuclear transfer (SCNT) derived viable sheep, “Dolly”, from transplanting an adult somatic cell into an enucleated sheep oocyte. Following this breakthrough, theoretically, it seems to be a promising technology to produce unlimited numbers of genetic copies from an adult animal or a fetus; and so far many species including farm, pet and endangered animals have been cloned using this technology. Obtaining a viable offspring through this technology still imposes a great challenge for researchers due to its relatively low success rate and in some instances compromised health status of offspring. At present, cloning is carried out for biomedical and research purposes, and agriculture production.

Method: The standard SCNT technique in most of the laboratories follows some common steps such as oocyte maturation, removal of cumulus cells from oocytes, enucleation, transplantation of the somatic cell into the perivitelline space of the oocyte, electrofusion of the cell couplet, activation of the embryo and in vitro embryo culture. When the embryos develop to blastocyst, they are transplanted into the uteri of estrus-synchronized surrogate animals 6-7 day post estrus. In most cases, the oocytes are collected from abattoir ovaries using the slicing method and undergo IVM. Some laboratories that conduct SCNT programs preferred in vivo derived oocytes either following ovulation or LOPU. This may be due to the decreased availability of goat abattoirs in different countries or the higher success rates from in vivo matured oocytes. Following maturation, the cumulus cells are removed by vortexing in 3% Sodium citrate medium [27] for 1-2 minute or in 0.01% hyaluronidase. Cumulus –free oocytes with first polar body extrusion are selected and after a short exposure to cytochalasin B and Hoechst stain, enucleation is carried out in a micromanipulator equipped with UV light. Following oocyte enucleation, cell cycle-synchronized donor cells are transplanted into the perivitelline space of the oocytes in a manner that both cell membranes attach firmly to each other. The cellcouplets are then electrofused in fusion medium. One hour following fusion, cell-couplets are activated by culturing in medium. Recent studies in our laboratory revealed that a greater incidence of chromosomal abnormalities occur when SCNT embryos are cultured in some medium. After activation, embryos are cultured in IVC medium and are transferred surgically (small animals) / nonsurgically (in large animals) into uteri of estrus –synchronized animals. 

Fig: Cloning by SCNT technique

 

Artificially splitting a single embryo at a very early stage of development.  In the natural process this would create twins. However, because this is done at an early stage and there are usually less than eight cells you can only make a few clones.  Both the nuclear genes and mitochondria genes would be identical.

Several large studies have now shown that the embryo does not transmit infectious diseases. In fact, the Research Subcommittee of HASAC, within the IETS, has categorised disease agents based on the risk of transmission with embryo (Stringfellow, et al 2004). Category 1 comprises diseases or disease agents for which sufficient evidence has accrued to show that the risk of transmission is negligible, provided that embryos are properly handled between collection and transfer. Proper handling includes the following:

Category 2, 3 and 4 diseases are those for which less research information has been generated. However, it should be noted that none of the infectious diseases studied has been transmitted by in vivo-produced bovine embryos, provided embryo handling procedures were followed correctly (Singh, 1985). Consequently, it has been suggested that embryo transfer be used to salvage genetic material in the event of a disease outbreak (Wrathall, et al 2004), whichcould be a useful alternative in establishing disease-free herds.

A prominent area of application of ART in combination with recombinant DNA technology research is the development of transgenic animals through genetic engineering. Transgenic animals are produced by introducing an isolated DNA fragment into an embryo so that the resulting animal will express a desired trait. Transgenic animals may be generated by the introduction of foreign DNA obtained through animals of the same species, animals of different species, microbes, humans, cells, and in vitro nucleic acid synthesis. 

Before going to details of the method one have to know about the stem cell. Several adjectives are used to describe the developmental potential of stem cells; that is, the number of different kinds of differentiated cell that they can become.

A. Totipotent cells:  In mammals, totipotent cells have the potential to become

The only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage (as shown by the ability of mammals to produce identical twins, triplets, etc.). In mammals, the expression totipotent stem cells is a misnomer — totipotent cells cannot make more of themselves.

B Pluripotent stem cells:  These are true stem cells, with the potential to make any differentiated cell in the body (but probably not those of the placenta which is derived from the trophoblast).

Three types of pluripotent stem cells occur naturally:

All three of these types of pluripotent stem cells

Using genetic manipulation in the laboratory, pluripotent stem cells can now be generated from differentiated cells. These induced pluripotent stem cells (iPSCs) are described below.

C. Multipotent stem cells:  These are true stem cells but can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. Multipotent stem cells are found in adult animals; perhaps most organs in the body (e.g., brain, liver, lungs) contain them where they can replace dead or damaged cells.

Transformation of embryonic stem cells:  DNA is introduced into a eukaryotic cell by a variety of techniques, such as transformation, injection, viral infection, or bombardment with DNA-coated tungsten particles (Figure ****). As we know that, when exogenously added DNA that is originally from that organism inserts into the genome, it can either replace the resident gene or insert ectopically. If the DNA is a transgene from another species, it inserts ectopically (Vectors that replicate autonomously in eukaryotic cells are rare; so, in most cases, chromosomal integration is the route followed.). 

Fig: Different ways of introducing foreign DNA into a cell. (Adopted from “An Introduction to Genetic Analysis. 7^th edition. Griffiths AJF, Miller JH, Suzuki DT. New York: W. H. Freeman; 2000.)

Step III:  Select for successfully transformed cells.

Step IV:  Inject these cells into the inner cell mass (ICM) of mouse blastocysts.

Step V:  Embryo transfer

Step VI:  Test her offspring:  Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.

Step VI:  Establish a transgenic strain

Fig: Diagrammatic presentation of production of transgenic animal.

Step I: Preparation of desired DNA as in Method 1 (Step I - StepIII).

There is several promising application of cloning, transgenics and recombinant DNA technology for human welfare through expression of desired characteristics in animal products like meat, milk etc. Some of these techniques are-

 

Controlled Breeding Programs in farm animals: This is achieved through application of exogenous substances (hormone / modification of photoperiod) to manipulate the cyclicity of female animals or birds. The hormone commonly uses for controlled breeding programme GnRH, FSH, Prostaglandin etc and modification of photoperiod (use of light) to stimulate the pineal gland activity commonly applied in seasonal breeding animal and birds.

 Potential Benefits of Controlled Breeding Programs

The term "estrous" refers to the point of female sexual excitement in mammals which causes ovulation. This period is commonly referred to as heat. Estrous synchronisation is the process of targeting female mammals to come to heat within a short time frame and this is achieved through the use of one or more hormones. GnRH and Prostaglandin F2 are two hormones used in the protocols during oestrus synchronisation. The synchronization of the estrous cycle is often used in order to decrease the costs for Artificial Insemination or feeding a bull by reducing the period in which it takes for all cows to be in heat and fall pregnant.

Progesterone Releasing Intra-vaginal Device (PRID): The PRID is a sponge that is inserted into the vagina of a cow to stop the natural estrous cycle (for it acts as a corpus luteum), because progesterone is the hormone that signals the body to stop the cycle because fertilisation has occurred. When the sponge is removed the cycle restarts.

Fertility medication, better known as fertility drugs, is drugs which enhance reproductive fertility. In female animals, fertility medication is used to stimulate follicle development. Agents that enhance ovarian activity can be classified as Gonadotropin releasing hormone and Gonadotropins.