Editing Przewalski's horse (section)

===Assisted reproduction and cloning===
[[File:Przewalski 26-9-2004-3.jpg|thumb|right|Przewalski's horses]]
In the earlier decades of captivity, the insular breeding by individual zoos led to inbreeding and reduced fertility. In 1979, several American zoos began a collaborative breeding-exchange program to maximize genetic diversity.<ref name=BC8204/> Recent advances in equine reproductive science have also been used to preserve and expand the gene pool. Scientists at the [[Smithsonian Institution]]'s [[National Zoological Park (United States)|National Zoo]] successfully reversed a [[vasectomy]] on a Przewalski horse in 2007—the first operation of its kind on this species, and possibly the first ever on any endangered species. While normally, a vasectomy may be performed on an endangered animal under limited circumstances, particularly if an individual has already produced many offspring and its genes are overrepresented in the population, scientists realized the animal in question was one of the most genetically valuable Przewalski's horses in the North American breeding program.<ref name=AP080617/> The first birth by [[artificial insemination]] occurred on 27 July 2013 at the [[Smithsonian Conservation Biology Institute]].<ref name=PHAI/><ref>{{cite magazine |title=First Przewalski's Horse Born Via Artificial Insemination |date=5 August 2013 |url=http://news.nationalgeographic.com/news/2013/13/130805-przewalski-horse-born-artificial-insemination-animal-science/ |archive-url=https://web.archive.org/web/20130807120240/http://news.nationalgeographic.com/news/2013/13/130805-przewalski-horse-born-artificial-insemination-animal-science/ |url-status=dead |archive-date=7 August 2013 |magazine=National Geographic |access-date=14 August 2013 |last=Shenk |first=Emily |df=dmy-all}}</ref>

In 2020, the first cloned Przewalski's horse was born, the result of a collaboration between [[San Diego Zoo Global]], [[ViaGen Pets|ViaGen Equine]] and [[Revive & Restore]].<ref name=":0">{{Cite web|title=Przewalski's Horse (Takhi) Project {{!}} Revive & Restore|url=https://reviverestore.org/projects/przewalskis-horse/|access-date=2020-11-15|language=en-US|archive-date=8 September 2020|archive-url=https://web.archive.org/web/20200908050955/https://reviverestore.org/projects/przewalskis-horse/|url-status=live}}</ref> The [[cloning]] was carried out by [[somatic cell nuclear transfer]] (SCNT), whereby a viable [[embryo]] is created by transplanting the [[DNA]]-containing [[Cell nucleus|nucleus]] of a [[somatic cell]] into an immature egg cell ([[oocyte]]) that has had its nucleus removed, producing offspring genetically identical to the somatic cell donor.<ref>{{Cite journal|last1=Tian|first1=X. Cindy|last2=Kubota|first2=Chikara|last3=Enright|first3=Brian|last4=Yang|first4=Xiangzhong|date=2003-11-13|title=Cloning animals by somatic cell nuclear transfer – biological factors|url= |journal=Reproductive Biology and Endocrinology|volume=1|issue=1|pages=98|doi=10.1186/1477-7827-1-98|issn=1477-7827|pmc=521203|pmid=14614770 |doi-access=free }}</ref> Since the oocyte used was from a domestic horse, this was an example of interspecies SCNT.<ref>{{Cite journal|last1=Lagutina|first1=Irina|last2=Fulka|first2=Helena|last3=Lazzari|first3=Giovanna|last4=Galli|first4=Cesare|date=October 2013|title=Interspecies Somatic Cell Nuclear Transfer: Advancements and Problems|journal=Cellular Reprogramming|volume=15|issue=5|pages=374–384|doi=10.1089/cell.2013.0036|issn=2152-4971|pmc=3787369|pmid=24033141}}</ref>

The somatic cell donor was a Przewalski horse stallion named Kuporovic, born in the UK in 1975 and relocated three years later to the US, where he died in 1998. Due to concerns over the loss of [[genetic variation]] in the captive Przewalski's horse population, and in anticipation of the development of new cloning techniques, tissue from the stallion was [[cryopreserved]] at the San Diego Zoo's [[Frozen zoo|Frozen Zoo]]. Breeding of this individual in the 1980s had already substantially increased the genetic diversity of the captive population after he was discovered to have more unique [[allele]]s than any other horse living at the time, including otherwise lost genetic material from two of the original captive founders.<ref name=":0" /> To produce the clone, frozen skin [[fibroblast]]s were thawed, and grown in [[cell culture]].<ref name=":1">{{Cite web|title=About Cloning {{!}} Revive & Restore|url=https://reviverestore.org/projects/przewalskis-horse/about-cloning/|access-date=2020-11-15|language=en-US|archive-date=30 November 2020|archive-url=https://web.archive.org/web/20201130143403/https://reviverestore.org/projects/przewalskis-horse/about-cloning/|url-status=live}}</ref> An oocyte was collected from a domestic horse, and its nucleus replaced by a nucleus collected from a cultured Przewalski's horse fibroblast. The resulting embryo was induced to begin division. It was cultured until it reached the [[blastocyst]] stage, then implanted into a domestic horse [[Surrogacy|surrogate]] mare,<ref name=":1" /> which carried the embryo to term and delivered a foal with the Przewalski horse DNA of the long-deceased stallion.

The cloned horse was named Kurt, after Dr. [[Kurt Benirschke]], a [[geneticist]] who developed the idea of cryopreserving genetic material from species considered to be endangered. His ideas led to creating the Frozen Zoo as a genetic library.<ref>{{Cite web|title=Kurt Benirschke (1924-) {{!}} The Embryo Project Encyclopedia|url=https://embryo.asu.edu/pages/kurt-benirschke-1924|access-date=2020-11-15|website=embryo.asu.edu|archive-date=30 October 2020|archive-url=https://web.archive.org/web/20201030173303/https://embryo.asu.edu/pages/kurt-benirschke-1924|url-status=live}}</ref> In 2021, Kurt was relocated to the breeding herd at the [[San Diego Zoo Safari Park]].<ref>{{cite web |last1=Beale |first1=Mel |title=Why a cloned foal born at a USA zoo is key to the survival of his endangered breed |url=https://www.yourhorse.co.uk/news/cloned-foal-key-survival-endangered-breed/ |website=Your Horse |language=en |date=10 June 2021}}</ref><ref name="SDZoo 2022">{{cite web |url= https://stories.sandiegozoo.org/2022/10/20/a-clone-and-his-mentor/ |title= A Clone and His Mentor |author= <!--Not stated--> |date= 20 October 2022 |website= sandiegozoo.org |publisher= [[San Diego Zoo]] |access-date= 29 February 2024 |archive-date= 29 February 2024 |archive-url= https://web.archive.org/web/20240229234821/https://stories.sandiegozoo.org/2022/10/20/a-clone-and-his-mentor/ |url-status= dead }}</ref> In order to integrate him into the existing herd, Kurt was partnered with a young female named Holly, a few months older than him, in order to allow him to learn the social and communication behaviors of wild Przewalski's horses. On reaching maturity at three to four years of age, Kurt is intended to become the breeder stallion for the San Diego Zoo herd to pass Kuporovic's genes into the larger captive Przewalski's horse population and thereby increase the genetic variation of the species.<ref name=":0" /><ref name="SDZoo 2022"/>

In 2023, a genetic twin of Kurt, named Ollie, was born from cloning with the help of the San Diego Zoo Global Frozen Zoo. It is the first reported case of any endangered species having more than one clone successfully produced. This individual eventually joins Kurt and Holly at the San Diego Zoo Safari Park.<ref>{{cite web|author=Black, A.|date=April 19, 2023|title=Second Przewalski horse born from cloning|url=https://www.cbs8.com/article/news/local/second-cloned-przewalski-horse/509-4c46e2f6-c583-4cd6-9b51-5065124f3e14|website=CBS News 8|access-date=April 20, 2023}}</ref><ref name="Novak et al">{{cite bioRxiv |biorxiv=10.1101/2023.12.20.572538 |title=Endangered Przewalski's horse, Equus przewalskii, cloned from historically cryopreserved cells |date= 9 January 2024 |last1=Novak |first1=Ben J. |last2=Ryder |first2=Oliver A. |last3=Houck |first3=Marlys L. |last4=Putnam |first4=Andrea S. |last5=Walker |first5=Kelcey |last6=Russell |first6=Lexie |last7=Russell |first7=Blake |last8=Walker |first8=Shawn |last9=Arenivas |first9=Sanaz Sadeghieh |last10=Aston |first10=Lauren |last11=Veneklasen |first11=Gregg |last12=Ivy |first12=Jamie A. |last13=Koepfli |first13=Klaus-Peter |last14=Rusnak |first14=Anna |last15=Simek |first15=Jaroslav |last16=Zhuk |first16=Anna |last17=Phelan |first17=Ryan }}</ref> Due to having been conceived through the transfer of a somatic cell nucleus into an egg cell obtained from a domestic horse donor, Kurt and Ollie both display the mitochondrial genome of domestic horses instead of belonging to a Przewlaski horse mithocondrial clade.<ref name="Novak et al"/> However, as [[Mitochondrial_DNA#Mitochondrial_inheritance|mitochondrial DNA is maternally inherited]], they will not pass on these domestic horse genes.