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SPOTLIGHT

Breeding Rhinos for the future The Artificial way

By felix patton

aptive breeding programmes have many decades about the husbandry and had some spectacular successes in reproductive needs of captive Black and 310 Csaving species and reintroducing Indian rhinos, when applied to White them into the wild. All rhino species Number of oestrous cycles rhinos, it has been unsuccessful. The are at risk from poaching, disease and non-reproducing White rhino main problem has been the absence of or natural disaster. Developing captive female in captivity can exhibit. erratic nature of oestrous cycle activity in populations is an essential safeguard to over half of the females in the European avoid extinction but breeding success has and North American Species Survival been limited. If a female rhino does not Program. get pregnant, her starts to develop White rhino females in the wild irreversible problems, such as cysts To date, captive reproduction in usually experience short intervals and tumours. Can the failure to become the White rhino has been poor. Despite between successive births, even as pregnant be overcome? the wealth of knowledge acquired over little as 18 months. This indicates that and lactation are the most common endocrine profile with possibly as few as 30 oestrous cycles per reproductive life span. Ultrasound technology for use in rhinoceros has been A reproducing female White rhino in used in well over 150 reproductive assessments in more captivity may produce up to nine calves. With a pregnancy of 16 months and than 70 rhinos with the result that the causes of poor subsequent lactation of approximately captive performance are now known. 12 months, the female exhibits only 90

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oestrous cycles during her reproductive vitro fertilisation (IVF), intracytoplasmic life span. With pregnancy and lactation injection (ICSI), and subsequent dominating the endocrine status, the 9 (ET) – together known oestrous cycle is a rare event during her Number of calves a as assisted reproduction technologies reproductive life. reproducing female white rhino (ARTs). Development of these A non-reproducing White rhino can produce in captivity. technologies for rhinos was initially female in captivity exhibits as many as slowed by the complicated structure 310 oestrous cycles. They display 90 of the reproductive organs. The main oestrous cycles by the age of 16 years. challenge to AI was to develop a special These non-reproductive periods of 10 to pass through the convoluted – 15 years in female rhinos were not than 70 rhinos with the result that the passage in the female's to deliver previously considered problematic. Now causes of poor captive performance are the sperm to the uterus. it has been found that 16 years old is now known. Female captive rhinos can The cervix is very firm and strongly the same age at which the first signs of develop uterine tumours, endometrial folded. This twisty route is the reason disease-caused alterations are detected in cysts and ovarian cysts. These conditions male rhinos spend about an hour and a the genital tract. occur more often as the animals age and half having sex because they must try to Ultrasound technology for use in when they have not achieved a pregnancy. fill up the cervix with sperm. rhinoceros has been used in well over This ‘asymmetric ageing process’ of the For egg collection, new equipment 150 reproductive assessments in more reproductive organs can be prevented and concepts had to be developed to get with the achievement of at least one to the , some 1.5 metres inside the pregnancy whether natural or by animal (compared to the 15 centimetres artificial means. Reproductive problems to a human ovary). A hollow needle, are relatively non-existent in the wild more than one metre long, fastened to an since females are either pregnant or ultrasonic head and inserted through the lactating during the greater part of their rectum is used. TOP LEFT: Checking a rhino reproductive life. This means that regular pregnancy using ultrasound. The needle can be viewed on an assessment of reproductive fitness and ultrasonic screen so the can early breeding are essential. TOP LEFT: Portion of a rhino cervix be seen, enabling the intestine to be With males, it has been found showing folds. accurately punctured, the needle pushed that unsuitable social systems impact towards the ovaries and the egg cells TOP RIGHT: Ultrasound image from a reproductive performance, resulting in sucked in. The method can reliably yield black rhino in Australia. half the captive population being sub- many egg cells. fertile or infertile. BELOW: Opened cervix with probe Well-established solutions to female through the convoluted cervical in domestic species and humans include artificial canal. FELIX PATTON is a rhino ecologist (AI), the in vitro production of embryos writing and broadcasting about the by means of (IVM), species from Africa and Europe. gamete intrafallopian transfer (GIFT), in

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Frozen sperm storage.

Artificial Insemination (AI) The Chimera Approach Non-surgical AI in White rhinos has been achieved using The body is made up of many hundreds of different types of fresh or cryopreserved semen. Additionally, rhino sperm cells, all of which come from a pool of stem cells in the early has been successfully sorted into high-purity X and Y embryo. Various types of stem cells give rise to the cells that chromosome–bearing populations. Critically small, captive carry out the specific functions of the body, such as skin, rhino populations could be boosted by using only X blood, muscle, and nerve cells. chromosome-bearing sperm, introduced by AI, to produce female offspring, enabling accelerated population growth. There are 3 types of stem cell that can be used, each with limitations:

i) Embryonic stem cells are a primitive type of cell that can be coaxed into developing into all of the types of cells (e.g. blood/heart/brain /nerve cells, etc). In the past, they have always been derived from embryos in a process that causes the latter’s death, and this has been considered unethical.

ii) Adult stem cells bear some similarities to embryonic stem cells but are limited in flexibility, and are only capable of developing into a few of the cell types. IVF, ICSI, Embryo Transfer In domestic species and humans, IVF (in vitro iii) Induced pluripotent stem cells are ordinary cells such fertilisation), ICSI (intracytoplasmic sperm injection) and as skin cells that are specially processed to exhibit some of ET (embryo transfer) are well- established techniques. the properties of embryonic stem cells without the ethical Where infertile, female rhinos have a history of and rejection problems. reproductive lesions, egg cell collection and in vitro ARTs represent the only option to preserve the female genetic Japanese scientists have developed a method to turn adult material and to contribute to the diversity of a population. cells, such as skin cells, into embryonic-like cells. In the future, it may (or may not) be possible to use the method to create For example, the Northern White rhino (Ceratotherium embryonic cells from a Northern White rhino and blend them simum cottoni) is now believed extinct in the wild. Only with the embryos of the Southern White rhino. The resulting eight remained in captivity and the females had mostly embryo is a "chimera", with a mixture of cells from both. Then become infertile so could not contribute to the genetic the hope would be that some of the resulting offspring, grown pool. Two males and two females were recently moved in surrogate Southern White rhino , would grow up to to an enclosed area in Kenya in the hope that they might produce the sperm and eggs of the Northern White rhino. breed in a more open environment (see SWARA 2010:01). An alternative/additional approach could be to use ARTs Scientists at Stanford University in California, USA have grown to obtain eggs from any of the living females, fertilise human eggs and sperm in the laboratory. The team used with male sperm in the laboratory and implant resulting stem cells taken from embryos but are hoping to use skin embryos in either Northern females or, as is more likely, cells in future. Once the method is proven it could be refined Southern White females (Ceratotherium simum simum) for rhinos as could other such scientific advances. These as surrogate mothers. Current rhino embryo production approaches would also permit the use of frozen cell samples techniques require refinement for consistent success. from dead individuals.

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MILESTONES IN THE USE OF ARTs IN RHINOS Glossary

was born in October 2008 at Budapest Artificial Insemination – the Zoo. The successful use of frozen semen introduction of sperm into the will enable reproduction experts to female by means other than sexual anaesthetise wild , collect semen intercourse. from them, and use the frozen sperm for Catheter - A hollow, flexible tube breeding offspring in international zoos. for insertion into a body cavity, duct, or vessel to allow the passage of fluids or distend a passageway. 2009 Chimera - an animal that has AI White rhino born in UK in 2009 The first White rhino to be born in Britain two or more different populations 2004 through AI arrived at Colchester Zoo using of genetically distinct cells that First conception from AI performed in the pioneering AI treatment developed at originated in different fertilised eggs. 2004 the IZW in Berlin. Chromosome - components in a Artificial insemination was first cell that contain genetic information. successfully used to conceive a - to preserve rhinoceros at Budapest Zoo, , biological tissue by freezing it and in 2004. holding it at very low temperatures. Embryo Transfer - placing an egg fertilised outside the womb into the 2005 female’s uterus or . First birth from AI performed in 2005 Endocrine – secretion of chemicals The world's first rhinoceros birth from directly into the blood stream. artificial insemination occurred at Endometrial – pertaining to the Budapest Zoo in January 2007. , which is the lining of the uterus. Gamete Intrafallopian Transfer 2006 2010 - a technique in which an egg is First Egg Cell Harvest in 2006 AI Indian rhino due in 2010 fertilised outside the body, then Scientists from the IZW, Berlin together Indian rhino female artificially inseminated implanted into one of a female’s with veterinarians from Australia in June 2009 using sperm collected four Fallopian tubes. successfully collected egg cells from an years previously and cryo-preserved at Intracytoplasmic Sperm Injection infertile, Black rhino. These were then Cincinnati Zoo’s CREW CryoBioBank before - a technique whereby a single sperm matured in a test-tube and fertilised being thawed and utilized, resulted in with rhino sperm but did not grow into the birth of a male calf in October 2010. can be injected directly into an egg in embryos. This shows that it is possible to produce an attempt to achieve fertilisation. offspring from behaviourally incompatible - a process Indian rhino pairs and allow new genetic by which egg cells are fertilised by 2008 material to be introduced in captive sperm outside the womb. First IVF Black Rhino Embryo in 2008 populations globally. In vitro Maturation – where eggs In June 2008, researchers successfully cells are matured in the laboratory created a Black rhino embryo which Collecting and banking sperm from rather than the ’s body. was cryopreserved until the technology genetically valuable male rhinos is an Lactation – the period following is developed to transfer it to an important step toward prolonging the birth during which milk is secreted. appropriate recipient. genetic life of founder animals and Oestrous Cycle – regular repeated preserving the genetic potential of males stages by which the body of a rhino that may otherwise never contribute to the female is prepared for reproduction. 2008 captive population. Ultrasound – high frequency sound First birth from use of frozen sperm in waves whose echoes can be turned 2008 ARTs for rhinos have been successful but into a picture. In June 2007, scientists from the IZW, the technologies need further refinement X chromosome – the sex Berlin artificially inseminated the to obtain greater consistency. With chromosome associated with female mother with cryopreserved sperm, appropriate funding the timescale should characteristics. which had been frozen for three years be short and a lifeline secured for those Y chromosome – the sex at minus 196 oC (minus 321 oF) in liquid rhino populations heading rapidly towards chromosome associated with male nitrogen. The world's first White rhino extinction. characteristics. to be conceived using frozen sperm

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