• INTRODUCTION
• Interspecies hybridization and hybrid
• Advantages of Interspecies hybridization
• Disadvantages of interspecies hybridization
• Hurdles in interspecies hybridization
• Isolation Mechanism
• Inability to invade oocyte wall by sperm
• Adverse effect of the Oviductal factors
• Polarity in Hybridization
• Large fetus size
• Unavailability of suitable foster mother
• Failure to Nuclear Reprogramming
• Mitochondrial/genomic DNA incompatibility
• Zygotic Genome Activation (ZGA) Failure
• Improper placenta formation and maternal immune response
• Interspecies Hybridization and Future Animal Production
• Conclusion

iar-23-2- f 3.jpg100.4KB

INTRODUCTION

 Interspecies hybridization (INH) is the crossing between two or more species (interspecies/interfamily/interorder/ interclass) to make offspring (hybrid) with new genomic constituents. Interspecies hybridization (INH) of the animals is an excellent research tool which can produce new animals with superior genetic makeup from different species/genera/family/order/class. However, huge rebuttals are given by some group who emphasize more the ethical stand point and fears that if this technology pervades would lead to offensive human-animal hybridization which could turn this planet into the "Planet of Apes". On the contrary, interspecies hybridization is being expected to solve many unexplained questions related to drug testing, vaccine production and regeneration of vital body parts such as liver, heart, and kidney (Beyhan et al., 2007). In addition to, interspecies hybridization could perfectly conserve endangered wild heritage of the world through rapid multiplication of small population using somatic cell nuclear transfer (SCNT)(Lanza et al., 2000; Dixon et al., 2007).

 Moreover, in case plants, interspecies plant hybridization have successfully developed plant varieties with desirable economic traits such as disease resistance, high production, sugar free fruits (useful to diabetic patients) (Sikka et al., 1978). On the other hand, interspecies hybridization in animals have very limited outcome till date with few exceptions (Lanza et al., 2000; Gomez et al., 2004; Folch et al., 2009). Progenies resulted from interspecies hybridization so far have almost showed infertility (Anderson, 1988; Mine et al., 2000) with the exception of sheep and goat cross which showed approximately double body weight than the average body weight of its dam and sire (Mine et al., 2000)

 Few exceptions which were successful through interspeicies hybridization were sheep and goat produced naturally (Handcock and Jacobs, 1966; Pinheiro et al., 1989; Mine et al., 2000) offspring from European mouflon (Ovis orientalis musimon) and domestic sheep (Ovis aries) (Loi et al., 2001), Bucardo (Capra pyrenaicapyrenaica) and goat (Capra hircus) (Folch et al., 2009), indian cattle (Bos indicus) and western cattle (Bos taurus) (Nahar et al., 1992), American bison (Bison bison) and European bison (Bison bonasus) or domestic cattle (Anderson, 1988; Kochhar et al., 2002), domestic cattle (Bos taurus) and banteng(Bos javanicus) or gaur (Bos gaurus) (Lanza et al., 200; Loi et al., 2011), and fertile progeny from swamp buffalo (Bubalus bubalis carabensis) and river buffalo (Bubalus bubalis bubalis) (Yang et al., 2010).

 Few success have also been seen in wild life i.e. hybridization of African wild cat (Felis sylvestrislybica)and domestic cat (Felis sylvestriscatus) (Gomez et al., 2004), Asian wolf (Canis lupus) and Dog (Canis domesticus) (Kim et al., 2007), Sand cat (Felis margarita) and Domestic cat (Felis sylvestriscatus) (Gomez et al., 2008).

 Beside this, there are so many success stories in hybridization experiments up to the production of blastocysts in vitro (Wanget al., 2007; Owiny et al., 2009; Soh et al., 2012). Currently, interspecies hybridization programs could follow highly specialized technologies such as interspecies somatic cell nuclear transfer (ISCNT) which avoids natural mating of interspeicies and in-vitro fertilization of oocytes with sperm from different species (Sansinena et al., 2005; Lu et al., 2005; Li et al., 2007). Nevertheless, we still face many obstacles and hurdles to successful interspecies hybridization. This review is done to point out the hurdles as well as their possible solutions. 

Interspecies hybridization and hybrid

 Species is a group of individuals that poses similar morphological and physiological characteristics and can produce fertile offspring through natural mating (Beyhan et al., 2007). On the other hand, interspecies hybridization is the act of mixing different species or varieties (normally within the same genus). Lately, scientists have been crossing the demarcation lined by the creator "The God" by crossing species of different genus/family/order and class and so on) of animals or plants and thus to produce hybrid. A hybrid is the resultant offspring from the mating of different species. The standard of success is different depending on taxonomic distances (how much closely related the species are) between donor animals. Hybrid offspring are already obtained from interspecies hybridization of closely related species (by crossing between goat and sheep, cattle and gaur, bison and cattle, horse and donkey, sand cat and domestic cat, Asian wolf and dog) (Lanza et al., 2000; Gomez et al., 2008; Folch et al., 2009) but most of them showed infertility without few exceptions (Anderson, 1988; Pinheiro et al., 1989 Mine et al., 2000). In continuation, blastocysts formation is considered as the highest success particularly for interfamily/order/class hybridization (Kim et al., 2004; Chang et al., 2004; Loi et al., 2011). Natural mating was only way of reproduction (both within species and interspecies) at the beginning of era and there were lots of barrier to natural mating between different species because of heterogeneity in body size, species behavior and physiological events (estrous cycle, gestation period, season of reproduction etc.) (Kochhar et al., 2002). Failure to fertilize oocytes by foreign sperm (through natural mating or incubation of oocyte and sperm together) was very common in past. But until today many different modern techniques have been made available to the reproductive biotechnologists to overcome obstacles in further easing the use of intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) technology (Kochhar et al., 2002; Selokar et al., 2011; Soh et al., 2012).

Advantages of Interspecies hybridization

Interspecies hybridization (INH) has shown it potential inplant breeding recently and in the past. Due to its success in plants many high yielding plant varieties and specific diseases resistant crop produce has enhanced humans livelihood, clothing and further facilitated medication (Sikka et al., 1978). On the contrary, in animal sciences, interspecies hybridization came up with new explorations and many unexplained obvious questions. INH is certainly a tool which is frequently used in biomedical sciences for e.g. development of embryonic stem cell lines of a particular species to another species (creation of human stem cell lines using cattle oocytes). Normally the desired outputs of this research are production of life saving vaccines, medicines and different novel therapies for human and animal diseases (Kim et al., 2004; Chang et al., 2004). Beside this, interspecies hybridization could also work to investigate the incompatibility between genomic and mitochondrial DNA from different species (Kim et al., 2004; Li et al., 2007). It might also help to find out actual gene sequence for production of specific protein responsible for particular gene expression or disease. In addition, this technology would also enhance conservation program for wild species through production of cloned embryos followed by cryopreservation or offspring production using a foster mother from other species (Wang et al., 2007; Li et al., 2007). It is also assumed the technology may open the door of new livestock strain development (with heavier weight, faster growth rate and better hardiness) by  combining genome from different species. For example, an interspecies sheep and goat hybrid have shown almost double body weight than his dam or sire (Mine et al., 2000; Beyhan et al., 2007). Furthermore, mule (interspecies hybrid of horse and donkey) is more efficient draft animal (take more load, more patience, better hardiness and lesser feed intake) than either horse or donkey and are very commonly used in Afghanistan and Pakistan for carrying load and other draft purposes (Proops et al., 2009). Mule duck is another fruit of interspecies hybridization which is originated from crossing between common duck (Anus platyrhinchus) and Muscovy duck (Cairinamoschota). The mule ducks are not fertile, neither male genital organ nor female genital organ is functional, this prevents the wastage of nutrients (nutrients are not required for genital organ development and reproductive cycle maintenance). Furthermore, growth rate of mule duck is always faster than either common duck or Muscovy duck and also shows heterosis (heavier body weight than the average weight of their parents). Moreover, mule duck meat does not have a bad smell like common duck meat; this meat is tenderer and has juiciness, so it is also widely accepted in the market and by consumers particularly in Taiwan where there are many mule duck production farm (Baeza et al., 2000; Wenlin and Xiujun., 2009). Another example of the advantage of interspecies hybrid is crossbreed cattle produced from crossing of European cattle (Bos taurus)and Indian cattle (Bos indicus). The hybrid cattle shows more body weight and better milk production ability than the indigenous (Indian) cattle with better disease resistance and heat tolerance over European cattle. It does not need delicious feed like European cattle and therefore, could easily cope with environmental and sociological conditions of Indian subcontinent (Nahar et al., 1992; Rokonuzzaman et al., 2009).

Disadvantages of interspecies hybridization

 In case of farm animal production, interspecies hybridization might have very few adverse impacts on production system and all of these are assumed like erosion of genetic diversity and opportunity of losing genetic purity at base population. In other words, production of high yielding and superior hybrid population will reduce demand of existing low yielding livestock to field level(farmers and stake holders). The commercial investor will rear new hybrid animals for better output and it might squeeze the genetic variation (all people will search potential genetic resources for better yield and better profit and monoculture production system might be practiced). Again, if production and marketing of hybrid animals is not monitored by proper authority then haphazard crossing and production system might pollute genetic purity of base population. And ultimate result would be poor adaptability to environmental hazards and if there were any disaster (new diseases) on animal industry then it would be very difficult to replace the infected population due to lack of genetic diversity. Another concern is dealt from ethical point of view and many people consider interspecies hybridization illegal. So, product of interspecies hybrid animal might not be accepted by all people or people of a particular region as genetically modified crops and fishes made controversy and not widely accepted (Curtis et al., 2004). In continuation, a group of scientist is alarming against interspecies hybridization and particularly between human-to-animal hybridization. It is apprehended that this type of hybridization program may create different types of ugly animals like chimpanzee. In case of human-animal hybridization, it might have some adverse impacts on human society as well. This could hamper normal social life like two-parents family might be vanished (young generation could be less interested to get married as marriage needs lots of binding, responsibilities and liabilities), cloned baby may feel shy to address him/herself to the society and relationship between parents and child might be loosen. Fertile married couple may also go for somatic cell nuclear transfer from people who are carrying superior genetic potential (famous and extraordinary people) and it may create genome selling tendency to celebrities (Ethics Committee ASRM, 2000).

Hurdles in interspecies hybridization

 Previously, it has been mentioned that there are lots of barriers to interspecies hybridization in animals that are creating difficulties in the success of this experiment. However, there are few aspects which are in reality the actual hurdles in the process have been discussed here.

Isolation Mechanism

 Isolation mechanisms are the natural obstacles that prevent the interspecies copulation and mating (McGovern, 1976). The most common obstacles during isolation mechanisms are anatomical differences, physiological heterogeneity and the geographic location differences of the concerned species (Kochhar et al., 2002). For example, cattle is at least four or five times larger than sheep or goat and they are different in morphological characteristics, behavioral pattern , breeding seasons and physiological processes (estrous cycle, estrous period and gestation length) that makes natural mating between cattle and sheep/goat impossible. As per the available knowledge and literature, even there is no evidence of showing sexual attraction to each other between cattle and goat or cattle and sheep. However, there are examples which have shown and proven to produce natural interspecies hybrid from goat and sheep. The cause may be the percentage of similarity in terms of anatomy and physiology is higher as compared to the dis-similarity between the last two species (Mine et al., 2000; Kochhar et al., 2002). On the contrary, cattle and buffalo have more or less similar anatomical and physiological characteristics such as the gestation period (buffalo have little large gestation period than cattle), estrous cycle and events occur during estrous period, the length, structure and size of uterus and oviducts are almost same and also food habit of these two species does not differ much (Drost et al, 1986; Abdel-Rahman, 2006). Furthermore, ancestors for these two species are same as per the karyotyping reports based on PCR-RFLP mitochondrial cytochrome b gene and species-specific repeat (SSR) gene technique. The karyotyping of that two species explored that diploid chromosome number of cattle and river buffalo are 60 and 50 respectively. But, the fundamental chromosome number of this two species are same (62), cattle has 58 acrocentric autosome and two bi-armed sex chromosome, on the other hand, buffalo has 38 acrocentric autosome, 10 bi-armed autosome and two bi-armed sex chromosome. It is believed that these five pairs bi-armed buffalo autosome is the result of the centric fusion translocation of the acrocentric cattle autosome (Iannuzzietal., 1987).

 In another experiment, PCR-RFLP mitochondrial cytochrome b gene and species-specific repeat (SSR) gene techniques were used, where the PCR amplification of the gene encoded cytochrome b gene resulted the same fragment (359-bp) in case of both cattle and buffalo, and also same restriction pattern fragments (190- and 169-bp) were found in both species when AluI digestion of the PCR fragment (359-bp) were performed (Abdel-Rahman, 2006). Furthermore, PCR amplification of the species-specific repeat (SSR) gene causes a 603-bp fragment for both cattle and buffalo. Moreover, the cattle autosome had similar banding pattern to the buffalo chromosome (Abdel- Rahman, 2006) these all are the indicator of their same origin. Being so much similarity between these species, there is no evidence to produce cattle-buffalo natural hybrids (Drost, 1984). This could be attributed to the fact that though these species live together in many rearing condition but may be due to the lack of attraction in showing libido to another species such interspecies hybrid was never encountered. In order to overcome the above mentioned restrictions/hurdles many alternative reproductive technologies were invented that makes interspecies hybridization experiments easier. These reproductive technologies are artificial insemination (AI), multiple ovulation and embryo transfer (MOET), ovum pick-up (OPU), in vitro maturation of ocytes (IVM), in vitro fertilization of oocytes (IVF) and in vitro culture (IVC) of embryos followed by transfer to a foster mother, handmade cloned (HMC), intracytoplasmic sperm injection (ICSI) and the most recent one is somatic cell nuclear transfer (SCNT) (Wang et al., 2007; Soh et al., 2012)

Inability to invade oocyte wall by sperm

This is a very common problem for both natural mating and in vitro fertilization (incubation of oocytes and sperm together) as it is mandatory to invade oocyte wall by sperm for successful infusion. However, before putting the oocyte to fertilize with sperm it is mandatory to properly isolating oocyte and collecting follicular fluid to be used for further culturing the oocytes (Fig.1). The entire process needs to done in aseptic conditions. Moreover, for collection of oocytes and follicular fluid one should know the structure and anatomy of the ovary in detail (Fig. 1). Later, droplets needs to be prepared for culturing of occytes and for further culturing the sperm with this oocytes (Fig. 2). Moreover, before performing invitro fertilization, oocytes needs to be graded thoroughly for collection of intact oocytes (Fig. 3). Oocytes are mostly invaded by the acrosome enzymes secreted from head of the sperm. This enzyme dilutes the cell junctions and zona pelucida and makes an entry path for sperm to the oocyte. But acrosome enzyme varies from species to species, enzyme from one species may not be able to dilute oocyte wall of other species with few exceptions(exception is found particularly for the closely related species like sheep and goat, cattle and buffalo) (Salvik and Fulka, 1992). This phenomenon acts as a great barrier to interspecies fertilization as this barrier is reasonable basically because it is one of the natural mechanisms which keep the genome of a species pure. For instance, when cattle are inseminated by buffalo spermatozoa and/or buffalo cows are inseminated by cattle spermatozoa they do not end up in pregnancy (Drost, 1984).

Fig. 1. Steps from ovary collection till the collection of follicular fluid and oocytes. A. Schematic presentation of ovary and its structure. B. Buffalo ovaries without corpus luteum. C. Buffalo ovaries with corpus luteum. D. Collection of follicular fluid from the graffian follicles by aspiration method using 2ml disposal syringe and added to Tissue culture media (TCM-199). E. Collection of oocytes for fertlization.

Fig. 2. Preparation of droplet culture dish. A. Demonstrationof droplet culture dish preparation using medium. B. Preparation of 600 ul droplet dish for washing of COCs. C. Preparation of 100 ul droplet dish for maturation of oocyte. D. Preparation of 100μl sperm containing droplet for fertilization of oocytes.

Fig. 3. Different grades of cumulous oocytes complexes (COCs). A. Normal oocytes Grade A (oocytes are completely surrounded by cumulous cells) and Grade B (oocytes are partially surrounded by cumulous cells) these two grades are used for fertilization. B. Abnormal oocytes Grade C (oocytes are almost not surrounded by cumulous cells) and D grade (degraded nucleus of oocytes) the last two grades are not used for further maturation and subsequent fertilization.

 To overcome this particular problem, intracytoplasmic sperm injection (ICSI) was seen as an excellent technique. In ICSI, the freeze dried sperm nuclear materials (sperm head) of desired species are injected to the donor oocytes through microinjection procedure followed by in vitro culture of fertilized oocytes to blastocyst stage and its subsequent transfer to a recipient mother or cryopreservation in liquid nitrogen. Today, ICSI is also been used for in vitro embryo production even it is a very comfortable technique for the production of test tube babies in humans (Li et al., 2003; Soh et al., 2012). The second alternative solution is interspecies somatic cell nuclear transfer (ISCNT). ISCNT is the technique considered exact as cloning of animals using a donor oocyte as well as uterus of different species and which does not require sperms (Beyhan et al., 2007; Selokar et al., 2011; Soh et al., 2012). In brief, in ISCNT, the donor oocyte is enucleated (the oocyte’s nuclear materials are removed) and later the somatic cell nuclear materials collected from a predefined animal are injected to the enucleated oocyte through microinjection. Finally, the resultant zygote is cultured up to the blastocyst stage in vitro and later transferred to a foster mother (normally to the uterus of oocyte donor) (Soh et al., 2012).

Adverse effect of the Oviductal factors

 When sperm is deposited to vagina of recipient animal either by natural mating or artificial insemination (AI), sperm passes the cervix of uterus and swims through oviduct to the site of fertilization. During this time, sperm acquire some extra capabilities that enhance the efficiency of fertilization. These events (sperm selection, maturation and capacitation) during transportation of sperm from the site of sperm deposition to the place of fertilization are very much essential for successful fertilization and subsequent development of zygote and the oviductal agents (components of oviductal fluid) patronize the whole process and the factors involved become oviductal factors. The common oviductal factors are hormones, growth factors and their receptors, enzymes and proteins including oviduct specific glycoprotein (this protein is produced at the time estrous cycle from the wall of oviduct and it regulates the penetration of oocyte by sperm and gets associated with zona pellucida (basically this protein prevent foreign sperm to penetrate the oocyte wall). Oviductal fluid is also responsible for efficient embryo development and protecting the embryos against oxidative stress (Salvik and Fulka., 1992; Aviles et al.,2010).

 The solution against this barrier is to avoid in vivo fertilization of oocytes, that is if oocytes are fertilized in vitro with desired spermatozoa, oviductal factors will not show adverse effect on sperm selection, maturation, capacitation and subsequent infusion of oocyte with sperm (as oviductal factors are absent during in vitro fertilization as these factors are secreted from wall of uterus and oviduct and only act when fertilization is done within live animal). For example, no fertilized oocyte was found after insemination of cows with ram spermatozoa, however, in vitro incubation of cattle oocytes with ram spermatozoa resulted a large number of fertilized oocytes with formation of both pronuclei (Salvik and Fulka, 1992). Two pronucleus formation was also found while fertilizing buffalo oocytes with cattle spermatozoa in one of our experiments (Fig. 4 and Table 1). Another experiment showed application of swim-up method for processing of sperm increases rate of interspecies fertilization in greater extent. That means good quality sperm and efficient separation method could reduce the adverse impact of oviductal fluid (Salvik and Fulka, 1992; Jameel, 2008). For this reason, during artificial insemination (AI) or in vitro fertilization (IVF) best quality sperm is taken using different sperm separation methods. Swim-up method is one of them. The other methods are simple wash method, Sephadex and glass wool columns and density gradient centrifugation. Among these methods, swim-up method (migration of sperm into the culture medium) is the oldest and most commonly practiced method (Jameel, 2008).

Fig. 4. Formation of pronucleus (arrow indicating pronucleus). A. No pronucleus formation (no penetration of oocyte with sperm). B. Formation of single pronucleus (either 2n or 1n). C. Double pronuclei formation (confirm fertilization of oocytes with sperm).

Table.1. In vitrofertilization of buffalo COCs using cattle spermatozoa based on pronuclei (PN) formation

Polarity in Hybridization

Polarity in hybridization and developmental potential is a very common phenomenon for interspecies crossing wherein successful hybridization could only be possible by using one combination of the species as another combination of the same species does not give same result and may result in zero success or limited success (Kochhar et al., 2002). This sort of polarity is found in reciprocal crosses such as sheep-goat and cattle-buffalo interspecies hybridization. Kochhar et al. (2002) proved polarity in interspecies hybridization of cattle and buffalo and demonstrated by exposing buffalo oocytes to cattle spermatozoa and cattle oocytes to buffalo spermatozoa. During this experiment Kochhar and his collegues (2002) found that buffalo oocytes had almost half efficiency to the efficiency of cattle oocytes to accept foreign spermatozoa. The observation revealed that buffalo oocytes have less efficiency to accept foreign spermatozoa as compared to the cattle oocytes which shows flexibility to fertilization and blastocyst development with alien spermatozoa (buffalo, gaur, banteng, sheep, goat and so) (Kochhar et al., 2002; Selokar et al., 2011). Polarity is also found in interspecies hybridization of sheep and goat. When goat are bred to sheep occur, but in most cases pregnancies are lost after few weeks without few exceptions (occasionally produce hybrid offspring). contrast, when sheep are bred to goat pregnancy does not occur or very seldom occur followed by subsequent abortion, this could be interpreted as inherent disqualification of the particular species to overcome block against interspecies pregnancy and subsequent development (Bunch et al., 1976; Anderson, 1988; Mine et al., 2000).

Large fetus size

 Complications during delivery of offspring are greatly related to abnormally large fetus. Each species has limited vaginal expansion and contraction ability (varies species to species) during giving birth. If fetus becomes extra-large, birth canal fails to deliver the offspring and causes dystocia. It may result the loss of either or both offspring and dam lives. Normally, in vitro production and embryo manipulation technique results a big proportion of abnormally large fetus (Walker et al., 1996). Moreover, interspecies hybridization between two unequal sized animals will result in an offspring most probably of intermediate size (average of dam and sire birth weight). So, fetus size could play an important role for interspecies hybridization and their delivery. For instance, if a bull is mated to a doe/ewe and pregnancy exists or if an in vitro produced cattle and goat hybrid embryo is transferred to a recipient doe and pregnancy sustain, then the resultant fetus will be of intermediate size between normal cattle fetus and goat/ewe fetus. That fetus will be larger than the usual doe/ewe fetus but smaller than the normal cattle fetus. However, birth canal of doe is not so large to sustain for delivery of a kid like a calf. So, it will create labor delivery (dystocia) or neonatal injury, labor breathing or death of either one or both of mother and offspring (Garry et al., 1996; Walker et al., 1996). The above problem of interspecies hybridization could be resolved by selecting larger species as mother. In other words, during hybridization between cattle and goat, one should use cattle for donation of oocyte and goat as sperm donor. Then the resultant offspring will come out easily from the uterus through a large birth canal. Another solution is by averting natural delivery and better to opt for caesarean section for the birth of the offspring (Kruip and Daas, 1997).

Unavailability of suitable foster mother

 Foster mother is the female animal used as uterus donor. That means this animal is used to carry the interspecies hybrid/cloned fetus. The selection of suitable recipient mother for interspecies hybrid embryo transplantation is not easy. Interspecies hybridization may not be possible in all reciprocal combination of species. Female individual from one species may carry hybrid pregnancy but the same pregnancy may not sustain to the foster mother from alternative species. That is polarity (differences in hybridization efficiency between reciprocal crosses) makes obstacle to the availability of recipient mother. For example, cattle oocytes exposed to buffalo spermatozoa result far better blastocysts yield than the buffalo oocytes exposed to cattle sperm (Kochharetal, 2002).

 For successful embryonic development and subsequent delivery, foster mother plays very important role as this individual is directly related to the developing embryo through nuclear reprogramming, genomic/mtDNA interaction, embryonic genome ctivation, providing physical support (through amniotic fluid and placenta formation) and nourishing embryo with nutrients (Wang et al., 2007). Beside this, maternal behavior of foster mother is also important for postnatal offspring as if the fster mother do not provide proper nursing to the offspring then it could result in death of the neonate. For example, chicken is a good foster mother for ducks, but duck are not good for chickens. Therefore, accurate selection of dam species (for best foster mother) is must and the knowledge of selection could be obtained from published literature or through repeated experimentations with all sorts of ermutation and combinations.

Failure to Nuclear Reprogramming

 The term nuclear reprogramming means setup of cell memory that has established during cell commitment and differentiation after somatic cell nuclear transfer. Each oocyte has potential to reprogram adult somatic cell nuclear materials to embryonic state that could produce new individual and the process of converting somatic cell nuclear materials to embryonic state is called nuclear reprogramming (Loi et al., 2011). Failure to the conversion of somatic cell nuclear material to totipotent state is a major barrier to the success of interspecies somatic cell nuclear transfer (ISCNT) (Li et al., 2007; Loi et al., 2011). In only few instances reprogramming compatibility was achieved but even in this the compatibility achieved is not 100% as the embryo produced still show many abnormalities including abnormal gene expression in both pre and post implantation period and even after birth (Chae et al., 2009; Xing et al., 2009).

Nuclear reprogramming efficiency could be improved by practicing some techniques and the potential tricks to enhance nuclear reprogramming are chromatin aremodeling agents (Tricostatin A, TSA), use of embryonic stem cells as nuclei donor and inactivation of Xchromosome in ISCNT embryos (Loi et al., 2011). Inoue et al (2010) reported the inactivation of one X hromosome in some females by a noncoding RNA which resulted in nine times more cloned offspring than females with both X chromosomes active. Furthermore, Kawamura et al. (2009) showed somatic cells containing tumor suppressor gene p53 have better nuclear reprogramming capacity when it is transferred to enucleated oocytes than somatic cells nuclei without p53 gene. So identification of specific genes related to nuclear reprogramming and their subsequent transfer to desired embryo might increase interspecies blastocyst development and offspring birth. 

Mitochondrial/genomic DNA incompatibility

 Genomic DNA is responsible for transcription, translocation and replication of mitochondrial DNA and mtDNA encodes various subunits of electron transfer chain that are responsible for ATP production. So integration between genomic DNA and mtDNA is must for normal cell proliferation, cell metabolism and overall development of embryo/fetus (Loi et al., 2011). Sometimes, same cell or individual have different mtDNA which is termed as mtDNA heteroplasmy (mixture of mtDNA in cytoplasm). Previously, it was assumed and thought that mtDNA heteroplasmy does not have impact on ISCNT. But Yan et al. (2010) studied on mtDNA heteroplasmy and observed some haplotypes of mtDNA and demonstrated that mtDNA enhances better development of cloned embryo. Normally, during ISCNT the genomic DNA is derived from the donor somatic cell and mtDNA is derived from the oocyte donor. The constituent of DNA varies from species to species. So, interaction between genomic DNA and mtDNA is not undesirable in interspecies hybrid embryos and it is found prominent in distinctly related species (Burgstaller et al., 2007).

 The suggested solution is insertion of more and more donor (nuclei donor) somatic cell mtDNA to recipient oocytes to enhance the heteroplasmy level up to normal cell physiology of nuclei donor (Loi et al., 2011).

Zygotic Genome Activation (ZGA) Failure

 Zygotic genome activation is the stage of embryonic development when all developmental processes gets under the control of zygotic genome and all the maternal influence gets degraded. This particular step is very important for smooth development and formation of blastocysts. As maternal end product interfere smooth growth of embryos, inter species/genus/family/order/ class hybrid embryos showed failure to zygotic genome activation in almost all research findings and the ultimately it causes embryonic developmental arrest. So activation of zygotic genome and elimination of maternal product is must for successful hybridization(Beyhan et al., 2007; Loi et al., 2011).

 Production of specific protein and delivery to desired zygote through microinjection might be an excellent solution to zygotic genome activation failure and avoid embryonic arrest. But it is necessary to know the actual sequence of genes which triggers zygotic genome activation (ZGA) and luckily the major genes responsible for ZGA have already been discovered (Loi et al., 2011).
Now, discovery of full gene sequence and production of specific protein followed by insertion to desired embryo could solve ZGA failure.

Improper placenta formation and maternal immune response

 Improper placenta formation means failure to proper attachment of fetus to uterine wall. The result of improper or no placenta formation is pregnancy failure (abortion). For example, ewes do not get pregnant usually when they are bred to buck naturally, but artificial insemination and embryo transfer technology assisted to establish hybrid pregnancies in ewes (Gustafson, 1993). However, the pregnancy is lost after several weeks of gestation with very occasional successful pregnancies (Bunch et al., 1976; Blum et al., 1997). Most probably, this pregnancy failure is the result of not or improper placenta formation (embryo does not able to attach with uterine wall) and some argue this to happen due maternal immune response (Loi et al., 2011). However, failure of sheep and goat hybridization is due to immune tolerance which is in contrast to the above argument. So, embryonic death of hybrid embryo is not the result of humoral immune attack and hybrid offspring should sustain to recipient mothers from both species without any abnormality (as they are tolerant to each other). Unfortunately, most research with sheep-goat hybridization showed abortion of developing embryos after several weeks. So, there is something another factor (s) that interfere the success and it might be the result of improper signaling at fetus/maternal interface (Blum et al., 1997; Oppenheim et al., 2001).

Interspecies Hybridization and Future Animal Production

Overcoming of the barriers in interspecies hybridization would certainly pave the way for new knowledge. Cattle are almost established as a universal foster mother because of its flexibility to accept and reprogram genetic material from a variety of species. By this time, production of human-to-cattle hybrid blastocysts and chicken-to-cattle hybrid blastocysts have already been reported and this would supposedly help in the establishment of human stem cell line and chicken stem cell line respectively without using oocyte from human and ova from chicken (Chang et al., 2004; Kim et al., 2004) and successful offspring production would just be a time factor. Certainly, these efforts for the production of interspecies hybrid might be a good solution to mitigate different fertility-sterility problems, genetic disorders in human and also for the invention of novel therapies. But, in animal industry this technology could make tremendous change in near future through the production of target specific animal products and combining superior genetic materials from different species. 

 For instance, cattle meat is well recognized for its taste, tenderness, juiciness and extraordinary demand and it is also the most expensive meat throughout the world. Moreover, cattle hide is superior to buffalo hide and is highly demanded by leather industries. In contrast, buffalo also have many superior traits over cattle like better digestibility to coarser feedstuff, heat tolerance, disease resistance, hardiness and also they are more gentle and easier to control (Valin et al., 1984; Franzolin, 1994; Sheikh et al., 2006). In future, an animal with new genomic constituents (combining traits of both cattle and buffalo) would perhaps enter into animal industry with better meat properties, hide quality, disease resistance, hardiness, heat tolerance, digestibility to coarse feedstuff, behavioral pattern and so on.

 Again, goat milk is anti-allergic, anti-carcinogenic, enriched with micro nutrients and has other medicinal values. Goat milk is an excellent medicine for asthmatic patients and cheese of goat milk is very delicious and demandable food. In addition, goat has larger litter size (average 2) than cattle. However, the small quantity milk productivity makes goat milk unavailable to people. On the other hand, cattle are recognized as number one dairy animal due to its high milk yielding ability (Jenness, 1980; Debski et al., 1987; Haenlein, 2004). Therefore, it is assumed that future dairy industry could have an animal that would produce large quantity milk like cattle but the milk constituent’s like goat and the animal would pose larger litter size than cattle.

 Sterility of interspecies hybrid is counted as a barrier to the sustainable interspecies hybrid animal production. But, sterility could be taken as an advantage to the future livestock production. As the male of almost all animal, bird and fish species show faster growth rate and heavier bodyweight than female and this is actually the result of sex hormone activity. The male hormone enhances to faster and higher growth rate over female hormone. Anyway, because of sterility none of the offspring would express their sexual behavior and the ultimate result might be equal growth rate and body weight gain as it is already established in fishes (Mair et al., 1995; Chakraborty and Banerjee, 2009; Chakraborty et al., 2011). Beside this, the resulted offspring could be a hybrid vigor (that is resulted offspring will show larger body weight than the average body weight of its parents) and it is found in one case of sheep-goat interspecies hybrid (Mine et al., 2000; Proops et al., 2009; Wenlin and Xiujun, 2009). Moreover, the sterile offspring will not be able to hamper the production goal through haphazard breeding.

 In case of multiplication of small population and introduction of exotic livestock to a particular area, interspecies hybridization could be used. For example, rain deer farming is common in Australia and New Zealand. But, this animal farm has not been established in Indian subcontinent yet. In future, if the policy maker thought to establish rain deer farm in Indian subcontinent, then it could be done by interspecies somatic cell nuclear transfer (ISCNT). That is transfer of somatic cell nuclear materials from rain deer to an enucleated oocyte of suitable foster mother could help to multiply and establish rain deer population in the desired area. There is an evidence to multiply mouflon (Ovis gmelini musimon) using domestic Corriedale sheep as foster mother in Argentina (Dixon et al., 2007).

 This technology could also be used for reproduction from deformed and sexually diseased animals. For example, an extraordinary bull is carrying organism for brucellosis then insemination of cows with semen from this bull will cause abortion and the only existing solution is culling of that bull. However, SCNT could help to reproduce from that bull using a foster mother (either from same species or another species).

Conclusion

 Plenty of barriers are already being emphasized and overcome and tremendous progress has occurred in the field of interspecies hybridization. Scientists discovered alternatives to natural mating, manual fertilization (fertilization of oocytes by sperm itself) and different genomic barriers. This technology is now not dependent on sperm donor; SCNT introduces dramatic turn to this technology. Foster mother problem might be solved very soon and cattle are supposed to be universal oocyte donor as successful because human-to-cattle and chicken-to-cattle blastocyst development have been already reported few years ago. Recently, a group of British scientists declared that they produced around 150 human-to-animal blastocyst and they are ready to invent new medicines and therapies. In continuation, geneticall modified crops and fishes are now readily available and some of them are contributing to food production and reduction of hunger. Hybrid chicken (commercial layer and broiler) and mule duck are now prime source of egg and meat to satisfy animal protein requirement of growing population. Hybrid of horse and donkey (mule) is successfully been used as draft power. However, the world hunger is growing day by day and we are not sufficient in food production yet, our food security is not so strong and many people still remain hungry every day. Due to which it becomes pertinent that it is high time to have new genomic constituents to enhance food production, protect diseases and lead better life. Now, the basic barrier is related to embryonic genome activation, nuclear reprogramming and interaction between genomic and mtDNA. Therefore, in future the  esearch emphasis should be given to overcome genomic barrier against successful interspecies hybrid animal production and enhance food security.