Read this article to learn about the most important approaches to artificial breeding of Animals!
i. Artificial insemination.
ii. Embryo transfer.
iii. In vitro fertilization.
iv. Embryo cloning.
Contents
i. Artificial Insemination:
As the male produces millions of sperms daily, the semen can be used to produce several off-springs. This is made possible by artificial insemination (Al) of females. For an effective Al to produce the desired results, the following aspects must be considered.
Semen Collection and Storage:
The semen ejaculate is collected, appropriately diluted and examined under microscope for the number of motile sperms. Normally, 0.2 ml of bull semen contains about 10 million motile sperms. The diluted semen can be used fresh within few days, or cryopreserved at -196°C in liquid nitrogen for long-term storage and transport.
Artificial insemination is done on standing animals through a technique, known a rectal palpation. Each semen ejaculate of a male bull, in theory, can be used to inseminate as many as 500 cows. Another advantage of Al is that the semen can be transported (in cryopreserved form) to different places/countries and used to develop superior animals.
Synchronization of Ovulation:
The female animals are inseminated after ovulation which can be detected by behavioural estrous. It is rather difficult to detect estrous, since it mostly occurs during night and lasts for only a few hours. The animal breeders would like to inseminate a large number of females simultaneously, so that the management becomes easy. For this purpose, the females are induced (synchronized) to ovulate at a set time. This is achieved by administration of progesterone and/or prostaglandins, which regulate ovulation cycle. Although total synchrony of ovulation is not possible, about 80% of the females could be made to respond by this approach.
Sperm Sexing:
The livestock industries prefer to have animals belonging to one sex. For instance, the dairy and beef industries demand more females than males. It is possible to produce the animals of desired sex through sperm sexing.
Sperms and ova contain half of the chromosomes of a somatic cell. Thus, an ovum contains autosomes and one X chromosome while the sperm contains autosomes and one Y chromosome. Sex is determined genetically by the sex chromosomes (X and Y). X chromosome is present in all the ova, whereas half of the sperm (of a semen ejaculate) possess X and the other half Y chromosomes.
The sex of the embryo is determined by the sperm (X or Y containing) that is successful in fertilization. Thus, if the embryo contains X chromosome, it is a female; while it is a male if it possesses Y chromosome. In the natural breeding, the sex ratio of progeny is close to 1:1.
It is possible to separate the sperms containing either X or Y chromosome, and use them selectively for the desired sex of the progeny. By employing a fluorescent dye (Hoechst 33342) and an instrument namely fluorescent activated cell sorter (FACS), two populations of sperms (X or Y chromosome containing) can be separated. Using this approach and in vitro fertilization technique has produced pre- sexed calves.
FACS separation of sperms is expensive and time (about 24 hours)-consuming. Many sperms may die before they are actually separated. Attempts are being made to develop better separation techniques of sperms.
ii. Embryo Transfer:
As the ruminant animal produces one egg at time, it can carry one pregnancy at a time. It is possible to increase the production of female animals by increasing the number of mature eggs from a given female, fertilize them and transfer (implant) the embryos (fertilized eggs) into a foster mother (recipient). The foster mother serves as an incubator, and does not make any genetic contribution to the offspring. Embryo transfer is a costly technique, and is selectively used for the production of animals of high genetic or economic valve.
Superovulation (Multiple Ovulations):
In the normal reproductive cycle of a non-pregnant female, one ovarian follicle (out of the 20 that develop) matures and ruptures, releasing one fertile egg at a time. The time of ovulation varies in different animals—21 days for cow and horse; 16 days for sheep and goat.
The circulating gonadotrophic hormone is closely associated with ovulation and release of egg. By increasing the concentration of this hormone, more ovarian follicles can be induced to ripen and produce more eggs. This process, known as superovulation or multiple ovulations may yield not less than 8-10 eggs at a time. Some animal breeders were successful in super ovulating animals to yield as many as 60 eggs at a time. This largely depends on the breed, nutrition and health of the animal, besides the environmental factors.
By administering prostaglandin F2a (PGF2a) and follicle stimulating hormone (FSH), estrous can be induced. Artificial insemination is carried out in the super ovulated females. Al is preferred to natural mating, since the purpose of superovulation is to genetically improve the progeny. As the eggs are fertilized, they undergo development to form embryos (Fig. 18.1).
Multiple Ovulation with Embryo Transfer (MOET):
Sometimes, the process of multiple ovulations (superovulation described above) and embryo transfer are considered together which is referred to as MOET. The embryos developed in the super ovulated animals (described above) are recovered after 6-8 days of insemination. In case of cattle, the recovery process is easy and can be done by using a catheter. For certain animals with smaller reproductive tract, surgical procedures may be needed to expose the oviduct and recover the embryos. These embryos are examined microscopically and identified.
The embryos are then transferred into a synchronized recipient (i.e. the foster mother) by using a procedure, which is comparable to artificial insemination. Alternately, the embryos can be frozen and stored for use at an appropriate time and place later. About 50-60% of pregnancy could be achieved in cattle with transferred embryos. Thus, a super ovulated female may result in 5-6 pregnancies.
The embryos may also be subjected to manipulations, as described below:
Embryo Splitting:
It is possible to increase the number of progenies (almost to double) by splitting the embryos. In addition, embryo splitting results in identical twins for various purposes (particularly genetic research). Embryo splitting techniques have been refined, and are routinely used these days. The embryos are suspended in a hypertonic (high osmotic) sucrose and bovine serum albumin (BSA) containing culture medium. This results in shrinking of embryo cells and settling of the embryos to the bottom of the container (usually a petridish) due to increased density.
Further, the embryos stick to the bottom due to electrostatic interaction of the negative charges on it (due to attachment of albumin) and the positive charges of the petridish. The inner cell mass (ICM) of the blastocyte can be bisected by using a surgical blade and a micromanipulation technique (with the help of an inverted microscope). Splitting of each embryo results in two equal halves.
As the embryos are split, each hemispherical cell mass reforms spheres. These split embryos can be transferred into the oviducts of synchronized recipients. The pregnancy rate with split embryos is about 5-10% less than the theoretical calculations. Thus, about 9 progenies (instead of only 5 in the original embryo transfer) can be developed through MOET. By MOET, it is possible to produce at least four identical offspring’s in animals at a time.
Embryo Biopsy:
Embryo biopsy involves the removal of a few cells from the embryo (mostly from the trophoblastic cells of the trophectoderm) for analysis to determine the sex. In recent years, this technique is becoming popular for the detection of genetic diseases. By using embryo biopsy, it is possible to stop the transfer of embryos with genetic abnormalities and undesirable traits.
Embryo Sexing:
Before the transfer or implantation of the embryo, the progeny sex can be determined. Embryo sexing is based on the principle of detecting the presence or absence of Y chromosome in the embryo biopsy cells. The absence of Y chromosome indicates that the embryo is a female.
In recent years, the presence DNA sequences specific to Y chromosome are being detected for embryo sexing. Another development is the use of polymerase chain reaction to amplify the DNA sequence (of Y chromosome) even from a single cell and determination the sex.
Limitations of Embryo Transfer:
i. The supply of embryo from super ovulated donors is limited.
ii. Freezing and thawing of embryos requires a lot of care to keep them functionally intact.
iii. Embryo transfer requires technical skill, besides high cost factor.
iii. In Vitro Fertilization:
The limitations of embryo transfer could be successfully overcome by using in vitro fertilization (IVF). IVF basically involves fertilization of oocytes of a female animal in the laboratory conditions under artificial conditions. This is in contrast to the natural fertilization, which occurs in the uterus. IVF is reasonably successful as it results in 70-80% of the fertilized eggs.
Through IVF technology, a large number of offspring’s can be produced from a single animal. Thus, a female animal, which normally produces 4-5 offspring’s in her lifetime, can produce as many as 50-20 offspring’s by employing IVF.
In vitro fertilization involves the following stages:
Oocyte Recovery:
During the course of estrous cycle, the ovarian follicles grow, get filled with fluid and become Graafian (antral) follicles. The oocytes from these follicls can be recovered by using laparoscopic surgery. It is possible to recover more number of oocytes from super ovulated donors.
In Vitro Maturation (IVM) of Oocytes:
The immature oocytes recovered in the above step, when incubated in vitro will result in mature oocytes (eggs). The occurrence of a large number of antral follicles throughout reproductive life in the ovaries of cattle and sheep are known.
It is theoretically possible to remove them by biopsy and induce oocyte maturation in vitro. This may ultimately result in the production of hundreds and thousands of eggs from a single female. However, the potential of in vitro maturation of oocytes is limited for various reasons-degeneration of follicles, unknown metabolic and hormonal conditions.
Fertilization of Eggs:
In vitro fertilization of the eggs is carried out by using semen obtained from a superior male animal. For IVF, the eggs are carried in small droplets (micro droplets) of culture medium. Each micro droplet usually carries about 10 eggs. A dose of sperm approximately one million cells per ml is adequate for IVF. The penetration of the sperm into the egg is facilitated by supplementing the medium with certain compounds (penicillamine, epinephrine etc.).
Embryo Culture:
The IVF embryos must be maintained in the in vitro conditions for a few days (about 7 days for sheep and goat, 8 days for cattle). This allows the development of embryos to blastocyte stage. Approximately, 60% of the IVF embryos in vitro culture can form blastocytes.
Implantation of Embryos:
The 7 or 8 day old embryos from the in vitro culture are implanted in the reproductive tract of the recipient female which acts as a foster mother or surrogate mother.
Limitations of IVF:
The pregnancy loss of IVF embryos, particularly during the first two months, is very high, although the reasons are not clearly known.
The following parameters, which may be considered as limitations of IVF, may contribute to fetal losses of IVF embryos:
i. Genetic defects in oocytes.
ii. Genetic defect in fertilizing sperms.
iii. Environmental mutagenesis.
iv. Inadequate supply of nutrients and hormones.
v. Exposure to toxic agents and free radicals.
In recent years, some improvements have been made in IVF to minimize the fetal losses.
iv. Embryo Cloning:
A clone represents a population of cells or organisms derived from a single ancestor cell.
Cloning basically means the production of identical copies of an individual:
It would be advantageous to increase the number of embryos from a particular embryo which possesses the desired characters. Two approaches are in use for embryo cloning.
1. Nuclear transfer.
2. Use of embryonic cells.