Role of Biotechnology in Assisted Reproduction

This article  following available assisted reproductive biotechnologies with current and/or potential applications to enhance reproductive efficiency and to obtain a large number of offspring from genetically superior/ infertile animals. In human, the same technology is used primarily to address infertility in couples and to treat certain diseases with stem cells.

• Artificial Insemination and Cryopreservation
• Multiple Ovulation and Embryo Transfer
• In Vitro Fertilization
• Sex Determination of Sperm or Embryos
• Embryo/ Oocyte Cryopreservation
• Nuclear Transfer or Cloning
• Transgenesis
• Stem Cells

Artificial insemination

No other technology in animal sciences has been so widely adopted globally as artificial insemination (AI), as it has revolutionized livestock productivity especially in cattle. Genetic constitution of an unknown animal can be replaced up to 50, 75 and 87.5% with elite animal in I, II and III generations, respectively. Progress in semen collection, evaluation, dilution and cryopreservation now enables use of a single bull simultaneously in several continents for breeding up to 100,000 cows in a year. This technology thus enables use of the very best bulls to serve a large cattle population. Also, each bull is able to produce a large number of daughters in a given time thus enhancing the efficiency of progeny testing of bulls. The high intensity and accuracy of selection arising from AI can lead to a four-fold increase in the rate of genetic improvement in dairy cattle as compared to that of natural service. Additionally, use of AI can reduce transmission of venereal diseases in a population and the need for farmers to maintain their own breeding males. Up to 75% or more conception rate has been reported in cattle following AI, however, success of AI depends on accurate heat detection, proper frozen semen handling and timely insemination by a trained inseminator.

In human infertile couples, if the female is fertile, AI is used to address the problem of impotentia couendi and oligozoospermia in males. In case of azoospermia in husband, sperm of donor (any other man) are used for AI.

AI is credited for providing the impetus for many other developments, which have had a profound impact on reproductive biotechnology. Studies of estrus detection and ovulation control which are essential for timely insemination, led to the development of embryo-transfer technology (ETT).

Multiple Ovulation and Embryo Transfer

ETT enables birth of multiple progeny from genetically superior (elite) females. To increase the number of embryos that can be recovered from elite females, the embryo donor is treated with gonadotropins to induce multiple ovulations or superovulation. Embryo donor is inseminated with high quality semen of a superior male. The fertilized embryos are collected from embryo donors by nonsurgical techniques. These genetically superior embryos are then transferred to genetically inferior but highly fertile females (embryo recipients) nonsurgically. Unlike AI which require about seven generations for nearly total genetic transformation, ETT enables birth of many genetically superior calves in one generation.

In cattle, generally 4-6 good quality embryos are collected after each superovlation and 50-60% recipients conceive following embryo transfer. Millions of calves have been produced using ETT  and in USA and Canada about 80% breeding bulls are produced using this technology.

In Vitro Fertilization

As multiple ovulation and embryo transfer has some limitations, production of embryos  in vitro (in the laboratory) is considered more efficient and economic. Immature oocytes (female eggs) are harvested from ovaries of elite embryo donors or infertile/ aged females. Ovum (egg) pick up (OPU) is a nonsurgical technique that uses ultrasound and a guided needle to aspirate immature oocytes from the ovaries. Once the immature oocytes have been removed from the ovary, they are matured, fertilized, and cultured in vitro for up to seven days until they develop to a stage that is suitable for transfer or freezing. Mostly embryos are transferred nonsurgically.

Though a cow normally produce only one viable oocyte during each estrus cycle (ovulation), up to 50 antral follicles exist on the ovary at any time of the estrus cycle. Using OPU, a valuable donor cow may potentially yield 15-20 oocytes each week (twice weekly collection of 7-10 oocytes per collection) or about 700-1000 oocytes/year/cow. Assuming a 30% blastocyst rate from those oocytes, and a 40% pregnancy rate, a cow may potentially offer 200-300 blastocysts or 80-120 pregnancies each year.

In human, baby born following in vitro maturation (IVM), in vitro fertilization (IVF) of oocytes and in vitro culture (IVC) of embryos in the laboratory and their subsequent transfer to donor or surrogate mother is generally called ‘Test-Tube Baby’. This technique is used in infertile couples where normal process of fertilization is impaired due to blockage of fallopian tube or other reasons.  If the husband of the lady is suffering with oligozoospermia or azoospermia, a process called intra-cytoplasmic sperm injection (ICSI) is being routinely used to inject even epididymal sperm or spermatids collected from testis, to accomplish IVF.

Sex Determination of Sperm or Embryos

Sexing of sperm/ embryo is considered to be one of the most desirable reproductive biotechnologies, as males are required for production of quality semen for the artificial insemination programme and meat industry, however, dairy farmers are interested in females only. Also, the number of females present determines reproduction potential of any herd. Therefore, pre-selection of females has got immense importance in preserving endangered breeds. Expression of foreign proteins in milk of livestock has further necessitated the pre selection of sex in favour of female.

Using a specific dye that binds to DNA (the Hoechst 33342 stain) and a flow cytometer/cell sorter, the DNA content of individual sperm is measured. In cattle, the X-chromosome bearing sperm (responsible for production of heifers) contains 3.8% (3.6-4.2%) more DNA than Y-chromosome bearing sperm, which produce bull calves. The X and Y chromosome bearing sperm can be sorted with the help of a cell sorter/ cell flow cytometer and the sexed semen is now made available commercially. With this technique, sex of calves can be predetermined with 90% accuracy. Following AI involving approximately 1,000 heifers, pregnancy rates following insemination with one million sexed, frozen sperm were reported to be 70% to 90% the rate of unsexed controls inseminated with 20 to 40 million sperm.  Sexed sperm are also used for breeding of donors after superovulation and IVF in cattle, where the requirement of sperm is very minimum (1-2 million).   

For sexing of embryos, application of Polymerase Chain Reaction (PCR) to amplify Y-chromosome specific (male specific) DNA sequences, directed by primers with a thermostable DNA polymerase from a small amount of DNA of one or more blastomeres (biopsed from an embryo) is the most reliable method. The accuracy of sexing by this method is reported to be about 95% and transfer of sexed embryos results in to 45-55% conception rate.

Embryo/ Oocyte Cryopreservation

Cryopreservation of embryo is an essential component in the commercial ET programme as not only the surplus embryos (more than the available recipients) can be frozen; it facilitates global movement of the complete animal as embryo.  Of the total embryos transferred globally, over 45% embryos are frozen. This technique can also be used for the conservation of endangered species and breeds, as unlike semen, complete genotypes can be conserved as embryos. Freezing of embryos is an established commercial practice especially in cattle.

In order to create ‘Oocyte Bank’, considerable progress has been made with cryopreservation of oocytes in the last 10 years. Viable oocytes have been recovered after freezing and thawing in various species of livestock and birth of progeny from embryos produced from cryopreserved oocytes have been reported in cattle. However, the present efficiency and reliability of using frozen thawed oocyte for generating offspring is, however, still lower than with cryopreserved embryos.

Nuclear Transfer or Cloning

A clone is a genetically identical animal, which can be produced either by embryo splitting (as occurs in nature)/ transfer of embryonic blastomeres (cells from undifferentiated cleavage stage embryos) or somatic cells (fibroblast, skin, heart, nerve etc) as donor nuclei. The promises behind this technology are, 1) the possibility to duplicate an unlimited number of copies of animals using somatic cell lines; 2) the possibility of genetic manipulations of the cell lines prior to cloning and 3) the feasibility of cloning a proven valuable animal.

The technique of somatic cell nuclear transfer involves culturing somatic cells from an appropriate tissue (fibroblasts) from the animal to be cloned. Nuclei from the cultured somatic cells are then microinjected into an enucleated oocyte obtained from another individual of the same or a closely related species. The nucleus from the somatic cell is reprogrammed to a pattern of gene expression suitable for directing normal development of the embryo. After further culture and development in vitro, the embryos are transferred to a recipient female resulting in the birth of live offspring. However, at present the efficiency of the cloning technology is low and more research is needed before this technology can be commercialized.

Transgenesis

The transgenesis or art of making transgenic farm animals has great potential in molecular breeding of farm animals, such as development of animals with high fecundity, higher fertility, disease resistance etc. Unfortunately, production of transgenic farm animals is very costly.  The pharmaceutical industry has been investing heavily in the production of transgenic farm animals as bioreactors or for xenotransplantation which has been keeping the transgenic research of farm animals ongoing.

Technology for producing transgenic mice has been well established and hundreds of transgenic animal facilities worldwide now offer commercial production of transgenic mice at a reasonable cost. However, contrarily, production of transgenic farm animals is not as simple as mice as there are only a handful of groups in the world who have a record of producing transgenic farm animals. Gene transfer in animals has been used for modifying the fat or protein synthesis in the mammary glands and numerous proteins have been produced in the mammary gland of transgenic sheep, goat, cattle, pig and rabbit.  Transfer of genes for imparting resistance to influenza virus in pig and cloning of the cow  from transgenic donor cells that express a Lysostaphin gene, rendering the cow resistant to mastitis have been reported.  Recently FDA approved use of anti-clotting  called ATryn, produced in the milk of genetically engineered goats. Compared to transgenic mice, the cost is high and the efficiency is low for making transgenic farm animals.

Stem Cells

Stem cells are unspecialized cells that renew themselves for long periods through cell division, these cells remain unspecialized until receives specific signals to become specialized. Under certain physiological or experimental conditions, they can be induced to become cells with specific functions like beating cells of heart muscle or insulin producing cells of pancreas, blood cells, neural cells etc. These cells can be utilized for the treatment of many chronic diseases which are considered incurable until now and screening of  toxins on varying range of cells. Stem cells can be obtained from an embryo or individual.

Embryonic stem cells are derived from the inner cell mass of the blastocyst. Adult or somatic stem cells are unspecialized cells found among specialized cells in a tissue or organ. These cells can renew itself and can differentiate to yield the major specialized cell types of the tissue or organs. Unlike embryonic stem cells, which are defined by their origin, the origin of the somatic cells in mature tissues is unknown. These adult stem cells seems to have the ability to differentiate into a number of different cell types (Haematopoietic stem cells- RBC, B-lymphocytes, T-lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages and platelets; stromal cells (mesenchymal stem cells) give rise to osteocytes (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells) and cells of tendons and if this differentiation can be controlled in the laboratory, these cells may be used for the therapy of several diseases.

In animals, embryonic stem cells of mice have been directed to differentiate in to oocytes  in culture suggesting that stem cell lines can be generated in cattle from genetically superior female to allow an unlimited number of oocytes to be derived. 

The spermatogonial stem cells are the only adult stem cells having the responsibility of transferring genes to next generations via the process of fertilization of ovum by the spermatozoa which are sequel of a sequence of events called spermatogenesis. Following the first successful transplantation of spermtogonial stem cells from donor to recipient resulting in donor-derived spermatogenesis and sperm production by the recipient a lot of research work has been done.  Cross-species spermatogonial transplantation from rats to mice and mice to rat has been achieved. Stem cells transplantation in pigs and goats was also successful. Heterologous transplant in the unrelated bull calves of different breeds has also been reported which raises the possibility of its use for commercial breeding as sperm arising from transplanted donor germ cells are capable of fertilization in vivo and in vitro. Potential application of this emerging technology are, 1) Surrogate production of spermatozoa, 2) Reduced time for progeny testing, 3) Production of transgenic animals, 4) Preservation of the germ-line of graded animals, 5) Production of exotic bull semen from indigenous bulls and 6) Conservation of endangered species.

1. AI will continue to be the important biotechnology for the dissemination of desired genes in the population of dairy animals for several decades to come. There is, however, need for the ‘value addition’ by improving the quality of semen and the services, increasing the coverage of breedable population, marker assisted selection of young bulls and their genetic evaluation.
2. Progress in the development of various reproductive biotechnologies have been phenomenal. Even at present level of efficiency, embryo technologies can significantly increase impact of superior genotype in the population.
3. Several of the third generation embryo technologies such as SCNT and Transgenic animal production may find important role in animal breeding and benefit dairy industry. Further intensive research is required in these areas and the challenge is out there for reproductive biotechnologists to face the above demanding requirement