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In 1996, the first mammal was successfully cloned, Dolly the sheep. In 2003, the first horse was successfully cloned. Since this time, many advancements have been made in the process of cloning as well as extensive research to improve the efficiency of cloning. The current efficiency of equine cloning is very low, ranging between 0.7% to 2.7%. Additionally, the pregnancy loss rate is at roughly 89% for cloned equine pregnancies. There are three main reasons that cloning horses has become an important topic: 1) to preserve genetics or animals that cannot reproduce like geldings or animals that suddenly die and have had incredibly successful careers or genetics 2) to preserve the genetics of exotic or endangered species, for example Przewalski’s Horse 3) to preserve the genetics of horses that serve as companion animals to some humans, giving them emotional fulfillment (Vanderwall et al., 2006). Studies ranging from in vivo versus in vitro to Traditional versus Zona Pellucida-Free (ZP Free), are helping to make improvements in countries across the world. There are different advantages to all cloning methods used and determining the efficiency of the methods is still in process. I believe that equine cloning is not the most efficient method to equine reproduction.
I believe a brief history of the cloning mammals and horses will help the understanding of the further discussed studies later in this paper. The first genetically identical horses were created in the 1980’s, however it was not by a cloning process, it was achieved through embryo splitting. Following this, the first cloned mammal, Dolly the sheep, was achieved through the traditional method of Somatic Cell Nuclear Transfer (SCNT). In 2003, the first equine clone was achieved however it was a mule, not a fully genetic horse. This was achieved through SCNT using a 45-day fetus cell, in vivo-matured oocytes and immediate oviducal embryo transfer. In 2010, a healthy cloned foal was created in Korea using ovum pick-up (OVU) derived oocytes (Gambini and Maserati, 2018).
The first thing that must take place in cloning an animal regardless of the cloning technique is getting the cells from the animal to be cloned. Typically, this is a section of tissue just under the skin. A veterinarian will prepare an area for surgery then take a biopsy to ship to the laboratory where the cloning will take place. The tissue arrives at the laboratory and is then minced and put into a culture in an incubator, where it will stay for roughly a week as the fibroblasts grow from the tissue onto the culture. The process of cloning is referred to as Somatic Cell Nuclear Transfer, or SCNT, there are multiple types of SCNT (Hinrichs, 2006). Majority of the studies revolved around cloning and SCNT show that there is a relatively low blastocyst and high pregnancy loss, with less than 5% resulting in healthy foals (Olivera et al., 2016). The blastocyst development rate for equine cloned embryos is only between 1% and 10% (Hinrichs, 2006). The “traditional” cloning process requires mature equine oocytes, they must be in the state ready for fertilization (or metaphase II of meiosis). Micromanipulation is the next step in the process. During micromanipulation, the chromosomes are removed from the oocyte via a fine pipette inserted into the oocyte. Once the chromatin has been the removed the oocyte becomes an ooplast. Next is recombination, this is typically done by placing the donor cell under the zona pellucida and using electric pulse to fuse the two. Activation must now take place, this is basically artificially doing the job of the sperm. This is achieved by increasing calcium in the oocyte, as the sperm would. The final step is transferring the activated recombined oocyte to the oviduct of a mare (Hinrichs, 2006).
Zona Pellucida Free (ZP-Free) SCNT is relatively new and has some advantages and disadvantages. An advantage of ZP-Free is that it can eliminate a few steps of the traditional SCNT method such as enucleation and cell fusion. Additionally, embryos using ZP-Free cloning can improve in vivoand in vitro embryo development by easily aggregating embryos (Gambini and Maserati, 2018). In research done by Dr. Cesare Galli, ZP-Free cloning method activation rate increased 90% from zona-enclosed methods with a cleavage range of 92.7% to 97.9%, as well as a 27.7% blastocyst production rate as opposed to a 9.77% rate in zona-enclosed. Additionally, Galli also obtained 13 pregnancies with a 23% foaling rate from 46 recipient mares and 118 embryos (Maserati and Mutto, 2016). Even so, the ZP-Free method also has some downfalls. It is a more difficult method due to the fact it is harder to transfer oocytes through the steps of cloning.
According to Olivero et al. (2016), “Using foetal [fetal] fibroblasts (FFs) rather than adult fibroblasts (AFs) in the horse led to an increase in blastocyst rates”. Additionally, recent information shows that pregnancy rates range as low as 9% for using adult fibroblasts (Gambini and Maserati, 2018). In a 2015 study by Choi et al. the live foal rate was only 26.66%, that is only four successful births from 15 pregnancies (Gambini and Maserati, 2018). Another study done by Olivera et al. (2016) concluded a 28.20% success rate with 22 foals from 78 pregnancies. A third study conducted by Gambini and Maserati (2018), had a 25% success rate with 1 foal born from 4 pregnancies. It seems as though, as concluded by Olivero et al. (2016), successful live births are at a very low success rate when using adult fibroblasts. This however is not an encouraging rate for the future of cloning. Most desires for cloning will come from seeing the success, attitude or performance of an animal which will likely be an adult animal at that point and thus lower the success percentage rate of a viable embryo and successful clone. It has been shown that around half (50%) of the live born cloned foals derived from adult fibroblasts have some form of pathology such as Neonatal Maladjustment Syndrome with enlarged umbilical cords or leg tendon contractures (Gambini and Maserati, 2018).
As stated earlier, Vanderwall et al. (2006) found the pregnancy loss rate of cloned equine pregnancies to be around 89%. It is believed that placental formulation abnormalities may play a role in the success or lack thereof in cloned equine pregnancies. These abnormalities are thought to possibly be caused by incomplete reprogramming of the somatic cell nucleus following the transfer to the recipient oocyte (Hinrichs, 2006). A study done by Vanderwall et al. (2006) looked more into what may be causing unsuccessful pregnancies by studying a group of horses and mules. Just as the normal cloning process the embryos were reconstructed, activated and surgically transferred to a recipient mare’s oviduct. They performed an initial ultrasound between day 12 and 16 of the study and then again, every seven to ten days until they reached day 60, at which point they then performed examinations every two to four weeks. During this study at each examination they looked at three things: “(1) the size and location of the embryonic vesicle(s); (2) the presence of an embryo proper within the vesicle; and (3) the presence of an embryonic heartbeat”. The study found that 33% (six of 18) of failed mule embryos and 14% (one of seven) of failed horse embryos, were small for their gestational age. Of the three successful mule pregnancies, all were of normal size for their gestational age. The second characteristic they looked at was the development of embryo proper, 43% of the cloned mule embryos failed to develop an embryo proper however this seemed to have no foresight into embryonic loss (Vanderwall et al., 2006).
In an attempt to improve efficiency of equine cloning, a study was done using ZP-Free cloning and differentiating the time between cell fusion and activation of the embryos. The results did in fact give some insight into a more efficient adjusted method for cloning equine. In this study focusing on the time between cell fusion and embryo activation, Olivera et al. (2016) obtained ovaries from slaughter houses in Argentina. Follicles were then opened to harvest cumulus-oocyte complexes (COG’s), these were then matured in vitro. The oocytes were then stripped of cumulus cells using hyaluronidase solution, then the zona pellucida was removed via incubated in pronase. The ZP-free oocytes were then enucleated. For the fibroblast part of this study, skin biopsies were obtained from 13 different horses. The ooplasts were then dropped over donor cells, the two stuck together and were then placed in a fusion medium where they were fused using electrofusion. The embryos were then activated, cultured for seven to eight days and then transferred using a transcervical method to recipient mares, aged four to ten years old. The effects of three different time periods between cell fusion and activation were observed. The three time periods included 1) less than one hour 2) one to two hours 3) two to three hours. There was a significant blastocyst rate increase as the time between fusion and activation increased. The two to three hour group showed an 11.5% increase, the one to two hour group showed a 5.6% increase and the less than one hour group showed 5.2% increase. Live foal births also increased tremendously in the longer fusion-to-activation periods. Of the 24 foals born from this study, 19 were from the two to three hour group, 5 from the one to two hour group and 0 from the less than one hour group (Olivera et al., 2016).
Another study done in 2014, looked at the transvaginal ultrasound guided follicle aspiration method for obtaining oocytes for equine SCNT. Lee et al. (2015) looked at the difference between short disposable needle (14-G) system and long double lumen needle (12-G) system in regards to retrieving oocytes. This study looked at the efficiency of preparation for cloning rather than the cloning itself through SCNT, nonetheless still an incredibly important step in the cloning process for equine. There was not a significant difference in the two needle retrieval methods, the 14-G had a 47.5% retrieval rate while the 12-G had a 35.0% retrieval rate. During this study, there was 26 SCNT embryos transferred to 13 mares. The cloning process was demonstrated the same as other studies using SCNT (Lee et al., 2015). One of the main reasons SCNT has not been studied more than it has is because obtaining the needed oocytes proves to be difficult, therefore making the retrieval process easier and more efficient can and will help to improve SCNT itself with more of an abundance of oocytes.
In a scientific prospective, equine cloning and cloning in general is still a relatively new topic of research. Although some research has been done, not nearly enough has been done to make cloning an efficient reproduction technique. Advancements and discoveries have been made in areas such as traditional and ZP-Free SCNT as well as adult versus fetal fibroblasts and more efficient oocyte retrieval processes. Within almost every study on equine cloning there are relatively low live birth or full-term percentages. I still believe that equine cloning is not currently an efficient method of reproduction for the equine industry. That does not go to say that many advancements are being made and that in the future it may in fact be a very efficient way to reproduce genetically high-quality horses to improve the industry.
- Gambini, A., and M. Maserati. 2018. A Journey through Horse Cloning. Reprod., Fertil. Dev. 30:8-17.
- Hinrichs, K. 2006. Equine Cloning. Vet. Clin. North Am.: Large Anim. Pract. 22:857-866.
- Lee, W., K. Song, I. Lee, H. Shin, B. Lee, S. Yeon, and G. Jang. 2015. Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system. J. Vet. Sci. 16: 509-516.
- Maserati, M., and A. Mutto. 2016. In Vitro Production of Equine Embryos and Cloning: Today’s Status. J. Equine. Vet. Sci. 41:42-50.
- Olivero, R., L. Moro, R. Jordan, C. Luzzani, S. Miriuka, M. Radrizzani, F. X. Donadeu, and G. Vichera. 2016. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors. PLoS One. 11.
- Vanderwall, D., G. Woods, J. Roser, D. Schlafer, D. Sellon, D. Tester, and K. White. 2006. Equine cloning: applications and outcomes. Reprod., Fertil. Dev. 18:91-98.
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