Reproductive Sciences

Reproductive Sciences

Reproductive Sciences

Most of the projects described involve collaborators from multiple states/countries and institutions who work in the field of animal and/or human reproductive medicine and sciences. A major objective for all of the programs, particularly for the international projects, is to transfer skills and technology so that our collaborators in the range countries of the species of interest can continue research and development on their own with the CCR staff serving in an advisory capacity after the initial training and preliminary studies.

The Reproductive Sciences Department, in collaboration with Omaha's Henry Doorly Zoo and Aquarium's veterinary and animal crews, has developed techniques such as artificial insemination and in vitro fertilization for many species and maintains a bank of more than 20,000 samples of frozen reproductive cells from over 50 species. This exceptional program helped produce the first test tube gaur and gorilla, and the first artificially inseminated and test tube tigers.  Currently, the program is working in conjunction with the University of Nebraska Medical Center to develop recombinant tiger hormones to increase the efficiency of these procedures in endangered felid species.  The program also produced the first artificially inseminated snake species, and continues leading assisted reproduction efforts in captive gorillas by utilizing sperm sex-sorting techniques in order to skew the sex ratio to produce more female offspring. Current efforts also include projects to develop assisted reproductive techniques in amphibian species.  All of these projects include an endocrine (hormone monitoring) component, to diagnose pregnancy or assess the results of novel treatment protocols by measuring reproductive hormone metabolites in urine or feces.

The Reproductive Sciences Department's long-term program in South Africa is applying reproductive technology to a variety of indigenous species that include elephants, lions, buffalo and a variety of antelope species. The team has developed a dramatic new technology that removes disease-causing organisms from reproductive material which has been issued patents in the US, Australia and New Zealand, and is patent-pending in Canada and Europe. This new technology will not only benefit wildlife populations, by reducing the risk of disease transmission, but also livestock and humans.

Tiger Assisted Reproduction

The Tiger Project has been a long-term program of the Reproductive Sciences Department that has focuses on all aspects of assisted reproductive technology including artificial insemination, semen freezing, in vitro embryo production (including fertilization by intracytoplasmic sperm injection), freezing and transfer, and the production of tiger-specific hormones for stimulating and synchronizing ovarian activity in tigers.

In-Vitro Embryo Production and Cryopreservation in Siberian Tigers

Studies have been conducted to test the effectiveness of pig (porcine) gonadotropins for repeatedly stimulating ovarian activity (follicle growth for oocyte or egg collection) in tigers. The porcine hormones are the most similar product commercially available. Sperm and in vitro produced tiger embryo procedures have been successful, however, pregnancy rates have been less than satisfactory using the porcine hormones to synchronize ovarian cycles. The success of the treatments are being examined by measuring reproductive hormone metabolites in the feces of the tigers.

Determining the Efficacy of a GnRH Implant for Performing Assisted Reproductive Techniques in Tigers

A continuation of previous laparoscopic assisted reproduction studies in tigers. In this case we want to laparoscope the tigers when they are in natural heats, artificially inseminate them and implant a synthetic hormone implant (deslorelin) to induce ovulation.

Sequencing Tiger-Specific Gonadotropins (specifically LH)

For use in ovarian stimulation and synchronization for cat embryo transfer. This project is a collaboration with the University of Nebraska Medical Center's Eppley Institute. Sequencing of the DNA section coding for tiger FSH and LH has been completed and transfection of cat kidney and Chinese hamster ovary cell lines is accomplished. Current efforts focus on propagation and testing of various cell line strains to find the most productive ones. The product will be collected in culture medium and concentrated by lyophilization will be tested for bioactivity in domestic cats.

Development of Novel and More Effective Techniques for Freezing (Vitrifying) Cat Embryos as a Model for Exotic Felids

Traditional methods for cryopreserving (freezing) embryos from livestock or humans can work with domestic cat embryos, but do not necessarily work for exotic felids such as tigers. A more recent technique, called vitrification, was found to be most effective for freezing cleavage stage embryos from in-vitro produced tiger embryos with an approximately 50% survival rate post-thawing.

Gaur Assisted Reproduction

The Gaur Project has been another long-term program of the Reproductive Sciences Department that has focuses on all aspects of assisted reproductive technology including artificial insemination; semen freezing, in vitro embryo production, freezing and interspecific embryo transfer.

Development of Artificial Insemination and Semen Freezing in Gaur

Adapting techniques used in domestic cattle, the first gaur calves were produced using fresh and frozen/thawed semen collected from live gaur bulls by rectal probe electrostimulation, or from sperm collected from gaur bulls after death.

Development of In Vitro Embryo Production and Interspecies Embryo Transfer in Gaur

Again, adapting techniques developed in domestic cattle, as many as six gaur calves have been produced by collecting oocytes (eggs) and fertilizing them in vitro then transferring them to recipient (surrogate) cows to carry to term. The following picture is of a gaur calf produced from oocytes collected from a gaur cow after her death, then fertilized using frozen/thawed gaur sperm from the cryobank and transferring the embryo to a domestic cow.

Gorilla Project

Funded by the Morris Animal Foundation, this project was designed as a management tool for the Association of Zoos and Aquarium's (AZA) Gorilla Species Survival Plan (SSP) in order to skew the current sex ration and produce more female offspring. Oocytes (unfertilized eggs) are collected from SSP-managed females for embryo production by the intracytoplasmic injection of sex-selected sperm (X-chromosome bearing) to produce female offspring. Oocyte collections and embryo transfers have been carried out at multiple institutions in the United States.

Assisted Reproduction in Snakes

Using the common corn snake as a model, researchers in the Reproductive Sciences Department were the first to ever report the successful birth of corn snake offspring of proven parentage using both fresh semen and semen cooled for three days prior to artificial insemination.  The goal of this research is to develop successful methods for artificial insemination using fresh, cooled or frozen/thawed sperm for critically endangered snake species such as the Jamaican boa.

Assisted Reproduction in Frogs

Using common species of temperate frogs (e.g., fire-bellied toads, leopard frogs and bull frogs) the Reproductive Sciences Department is contributing to the Omaha's Henry Doorly Zoo and Aquarium's commitment to the global efforts for the amphibian extinction crisis. Non-invasive methods are being tested to induce and extend the period of sperm release (spermiation) in seasonal frogs in order to enhance reproductive performance throughout any given year. Methods found to be effective in these model species will then be applied to more critically endangered amphibians such as the Mississippi gopher frog.

Hormone Monitoring

Non-Invasive Diagnosis of Pregnancy and Reproductive Disorders in a Variety of Species by Measuring Fecal Reproductive Hormone Metabolites

To diagnose pregnancy or determine if there is a reproductive disorder in a non-domesticated animal, multiple samples are required over an extended time period.  For that reason, blood sampling is not an option because this would require chemical immobilization, unless the animal has been trained to allow voluntary blood sampling.  Techniques have been developed to measure the metabolites of reproductive hormones in urine or feces, and this has proved to be an invaluable procedure to:

  • Determine seasonality for male/female introductions
  • Diagnose pregnancy and forecast parturition
  • Evaluate the effectiveness of assisted reproduction protocols such as exogenous hormone delivery and artificial inseminations
  • Evaluate fertility/infertility of individual animals.  These procedures assist in improving reproductive success and the well-being of animals in managed breeding programs.

Monitoring of Stress in Captive Female Okapi

Using urinary and fecal hormone analyses: This is a collaboration with Dr. Cynthia Bennett of the Dallas Zoo. The results may be useful for improving husbandry of captive okapi.

Reproductive Cryobank

Development of Optimal Methods for Cryopreserving Sperm in a Variety of Species

An ongoing project conducted opportunistically upon the death of an animal or with semen collected during a routine medical examination.  It is important to note that, as with tigers, wild or domestic animal counterparts do not always serve as model species for determining what treatment regimen should be used for freezing sperm.  Each species often requires refinement or modifications of existing protocols to be optimal for cryobanking semen.

Embryo Production by In-Vitro Oocyte Maturation, Fertilization and Culture and In-Vitro-Derived Embryo Cryopreservation in Domestic Cattle

These procedures are used as quality controls for the CCR In Vitro Fertilization Laboratory as well as for training of new students and personnel in the proper handling and processing of mammalian oocytes and the in-vitro production of embryos.  Since unfertilized oocytes can not be frozen in practically all species, it is necessary to produce embryos by in vitro fertilization since these generally can survive freezing and thawing.

Cryopreservation and Culture of Ovarian Tissue

In light of the fact that unfertilized oocytes can not be cryopreserved very successfully in any species, researchers have demonstrated in humans and laboratory animals that ovarian tissue may be a more effective means of salvaging gametes from females after ovariectomy or death.  The tissues are frozen then thawed and cultured until the ovarian follicles grow to a point that oocytes can be recovered and fertilized to produce viable embryos.  These same procedures are being tested by the staff and students of the CCR Reproductive Sciences Department on model species such as domestic cats and cows, with the intention of eventually applying effective methods to their endangered species counterparts.  Below is an electron micrograph of a domestic cat ovarian follicle containing a maturing oocyte.

Omaha's Henry Doorly Zoo and Aquarium's CCR Genome Resource Bank

Currently, over 20,000 individual samples of sperm and embryos from over 40 species are in liquid nitrogen storage and monitored by a state-of-the-art computerized system.

International Projects

Worldwide

In-Vitro Embryo Production and Cryopreservation in Arabian Leopards

A collaboration between Omaha's Henry Doorly Zoo and Aquarium and the Breeding Centre for Endangered Arabian Wildlife, Sharjah, UAE, to apply techniques of assisted reproduction to the conservation of the critically endangered Arabian leopard in a range country.

In-Vitro Embryo Production and Cryopreservation in Brazilian Jaguars

A collaboration between the Zoo, Associacao ProCarnivoros, the University of Sao Paulo and several Brazilian zoos to develop and apply techniques of assisted reproduction in jaguar in the species range country.

Developing Optimal Methods for Cryopreserving In-Vivo-Derived Embryos and Semen Collected from Dromedary Camels

An ongoing program in collaboration with the Camel Research Institute in Dubai, UAE. An annual workshop is held with participants coming internationally to learn more about embryo collection, production and transfer in camels – a species that is the main source of milk, meat and fiber in many countries. The outcome of these studies could aid in the application of these techniques to endangered camelid species.

The International Embryo Transfer Society (IETS) Parent Committee on Companion Animals, Non-Domestic and Endangered Species (CANDES)

This committee was established in 2001, chaired by Dr. Naida Loskutoff, and whose activities have been sponsored by Omaha's Henry Doorly Zoo and Aquarium. This is the only known international forum for Reproductive Physiologists, Biologists and Technologists to discuss issues pertaining to assisted reproductive technologies in these species. The committee's mission statement is to serve as an informational resource pertaining to the successful application of embryo technologies in CANDES by:  

  • Assisting in the understanding of their unique reproductive biologies
  • Provide instruction on proven method of various reproductive biotechniques
  • Advise on the technical feasibility and realistic expections for advanced biotechniques based on previous scientific evidence
  • Develop strategies for reducing the risk of disease transmission in CANDES.

The IETS CANDES website is available at www.iets.org and click on the CANDES tab.

On the African Continent

A large portion of the Center for Conservation and Research efforts in Reproductive Sciences Department focuses on the African continent, specifically in South African and Kenya, ranging from research on wildlife, livestock as well as humans.  Since 1994, Dr. Loskutoff, her staff and students have regularly traveled to South Africa to work on a variety of projects.  Their base of operation has changed from centers at the Johannesburg Zoo, the Jubatus Cheetah Reserve in the Limpopo Province, and currently plans are being developed to establish a center of operation with the South African National Parks Board.  

Semen Decontamination Technology

Dr. Naida Loskutoff developed this novel technology to conserve and expand the genetic diversity of rare and endangered hoofstock in zoos by assisted reproductive technology rather than capturing and transporting live animals.  In order to expand the gene pool of a captive population of a rare African antelope species, a zoo would have to bring new breeders from the wild into the U. S. However, in order to protect our U.S. domestic livestock industry, the United States Department of Agriculture (USDA) requires a very long and costly quarantine process to make sure the animal does not bring in infectious diseases. Unfortunately Africa and much of Asia and South America are infected with viral diseases such as hoof and mouth disease and rhinderpest which could decimate our livestock industry.

It would be faster and less expensive to simply import frozen semen into the U.S. to artificially inseminate captive females. Unfortunately fresh and frozen semen can carry disease agents. Dr. Loskutoff's passion for reproduction led her to a novel idea. She first added an enzyme to a semen washing and filtration process she was developing. She then designed plastic ware that when used with the enzyme-washing-filtration process, separates healthy sperm cells cleaned from the infectious agents, thereby making the procedure fool-proof to do even a field lab. Because of this unique patented procedure, male animals do not need to be captured, transported and quarantined at great cost. Only the frozen processed semen needs to be transported. It is hoped that this original research will attract the attention of USDA regulatory officials and similar decision makers world wide.

In an interesting turn of events the process works equally well on human diseases such as HIV and hepatitis. The South African government (Medical Research Council) supported most of the human research on HIV and hepatitis which was conducted in South Africa. Clinical trials are now scheduled to begin as soon as possible in Europe. It is possible that a project designed to benefit rare and endangered hoofstock species in U.S. zoos may have very beneficial implications for humans and livestock as well.

In May, 2007 the U.S. Trademark and Patent Office approved the Omaha Zoo's request for a patent on this semen decontamination procedure.  The procedure is now being used in field trials with livestock and clinical trials in humans and has been given exclusive rights to a company for eventual marketing of a product that was never before available to human, livestock and wildlife programs.

Determining the Efficacy of the Novel Semen Disinfection Procedure for Eliminating HIV and Hepatitis B and C Viruses from the Semen of Infected Humans

Because of the high prevalence of sexually transmitted diseases such as HIV and hepatitis in South Africa, there was a great interest by clinicians and embryologists working at human fertility centers to test the novel decontamination procedure on the semen of infected men.  It was found that the procedure was not only effective in removing these viruses, but it was equally effective for a variety of other organisms (e.g., bacterial and other microbial agents) without any detrimental effects to sperm viability.  This research was funded mostly by the Medical Research Council in South Africa and plans are currently in place to establish centers to provide this treatment for couples seeking assisted conception.  The application of the procedure to humans will help regulatory officials internationally recognize the merit of the procedure for decontaminating semen which is hoped would eventually lead to a lessening of restrictions for the import/export of semen for use in assisted reproduction in livestock and wildlife.

Determining the Efficacy of a Novel Semen Decontamination Procedure for Indigenous African Cattle Breeds

Some of the initial experiments testing the efficacy of the semen decontamination procedure were also also conducted on indigenous breeds of African cattle (e.g., Ankole, see photograph below).  These experiments were designed not only to determine the effectiveness of the procedure for removing potential pathogens from bull semen (bacteria, viruses and various microbial agents) but to examine if the procedure had any detrimental effects on sperm after artificial insemination, which is a multi-million dollar industry worldwide for the genetic improvement of livestock.

Disease Management in Cape Buffalo through Assisted Reproduction

This field project in Kruger National Park in South Africa focuses on using the semen decontamination procedure to cryobank semen from diseased African buffalo for the long-term genetic management of the species.

Programs and Student Training

Programs

Programs in the Reproductive Sciences Department at the Bill and Berniece Grewcock Center for Conservation and Research at Omaha's Henry Doorly Zoo and Aquarium focus on the development of tools that will be of value in the long term conservation of genetic diversity in endangered species, including:

  • In vitro embryo production
  • Cryopreservation of gametes and embryos
  • Non-invasive hormone monitoring to diagnose pregnancy and determine the success of treatment protocols
  • Disinfection procedures for gametes and embryos prior to utilization in assisted reproduction (e.g., artificial insemination and embryo transfer)

Most of the projects described involve collaborators from multiple states/countries and institutions who work in the field of animal and/or human reproductive medicine and physiology. A major objective for all of the programs, particularly for the international projects, is to transfer skills and technology so that our collaborators in the range countries of the species of interest can continue research and development on their own with the Zoo's staff serving in an advisory capacity after the initial training and preliminary studies.

Omaha's Henry Doorly Zoo and Aquarium and Hillsdale College Student Training Program

Sponsored by Berniece Grewcock annually since 1998, this program was developed to give undergraduate students in the Biology Department at Hillsdale College (Michigan) an opportunity to participate in a variety of scientific disciplines at Omaha's Henry Doorly Zoo and Aquarium's Center for Conservation and Research and/or Omaha's Henry Doorly Zoo and Aquarium's program in South Africa.

In South Africa, the research included reproductive studies on species such as rhinos, buffalo, and a variety of antelope species and genetic studies on species such as lions, cheetahs and ground hornbills. Currently, the genetic research, supervised by Hillsdale College Professor Dr. Daniel York, has been focusing on the origins of the Chytrid fungus - the pathogen that has been identified in playing a major role in the global amphibian extinction crisis.

Publications

Vogler, B.R., Blevins, B.A., Goeritz, F., Hildebrant, T.B., and Dehnhard, M. (2009). Gonadal activity in male and female captive fossas (Cryptoprocta ferox) during the mating season. Reproduction in Domestic Animals 44:98-102.

Blevins, B.A., Armstrong, D.L. and Loskutoff, N.M. (2009) Tiger (Panthera tigris) ovarian stimulation: the effect of timing on exogenous hormone delivery. Reproduction, Fertility and Development 21(1):153.

Mattson, K.J., DeVries, A.T., Krebs, J. and Loskutoff, N.M. (2009) Cryopreservation of corn snake, Elaphe gutatta, semen.  Reproduction, Fertility and Development 21(1):161.

Pedersen, M.J., Watson, C.A., Blevins, B.A. and Loskutoff, N.M. (2009) Domestic cat (Felis catus) embryo cryopreservation: slow-cooling versus vitrification. Reproduction, Fertility and Development 21(1):163.

Aaltonen, J.T., Mattson, K.M. and Loskutoff, N.M. (2009) The effect of a novel recombinant protease treatment on bovine sperm fertilizing capacity and embryo development in vitro. Reproduction, Fertility and Development 21(1):104.

Blevins, B.A., de la Rey, M. and Loskutoff, N.M. (2008) Effect of density gradient centrifugation with trypsin on the fertilizing capability of bovine sperm in vivo. Reproduction, Fertility and Development (accepted; in press).

Mattson, K.J., Devlin, B.R. and Loskutoff, N.M. (2008) Comparison of a recombinant \ trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos. Theriogenology (accepted; in press).

Nascimento, J.M., Shi, L.Z., Meyers, S., Gagneux, P., Loskutoff, N.M., Botvinick, E.L. and Berns, M.W. (2008) The use of optical tweezers to study sperm competition and motility in primates. Journal of the Royal Society Interface 5:297-302.

Aaltonen, J.T., Bedows, E., Estes, K.A.,. Butnev, V.Y., Bousfield, F. and Loskutoff, N.M. (2008) Production of recombinant LH for use in tiger (Tigris altaica) assisted reproduction: a preliminary report. Reproduction, Fertilty and Development 20(1):159.

Blevins, B.A., de la Rey, M. and Loskutoff, N.M. (2008) Effect of density gradient centriguation with trypsin on the fertilizing capability of bovine sperm. Reproduction, Fertilty and Development 20(1):84.

Mattson, K.J., Devlin, B.R. and Loskutoff, N.M. (2008) Comparison of a recombinant trypsin versus the pig pancreatic extract on in vitro-produced bovine embryos. Reproduction, Fertilty and Development 20(1):184.

Mattson, K.J., DeVries, A., McGuire, S.M., Krebs, J., Louis, E.E. and Loskutoff, N.M. (2007) Successful artificial insemination in the corn snake, Elaphe gutatta, using fresh and cooled semen. Zoo Biology 26(5):363-370.

Blevins, B., Steenson, S. and Loskutoff, N.M. (2007) Effects of different trypsin sources and concentrations on the viability of bovine sperm pre- and post-cryopreservation. Reproduction, Fertility and Development 19(1):121.

Mattson, J.K., DeVries, A.T., McGuire, S.M., Krebs, J., Louis, E.E. and Loskutoff, N.M. (2007) Successful artificial insemination in the corn snake, Elaphe gutatta, using fresh and cooled semen. Reproduction, Fertility and Development 19(1):240.

Chatfield, J., Zhang, L., Ramey, J., Bowsher, T., Loskutoff, N.M. and O?Neill, K. (2006) Resolution of a prolactinoma in a western lowland gorilla (Gorilla gorilla gorilla). Journal of Zoo and Wildlife Medicine 37(4):565-566.

Spindler, R.E., Crichton, E., Agca, Y., Loskutoff, N.M., Critser, J., Gardner, D.K. and Wildt, D.E. (2006) Improved felid embryo development by group culture is modified but not lost with heterospecific companions. Theriogenology 66:82-92.

Loskutoff, N.M. (2006). Proceedings of the Third International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Companion Animals, Non-Domestic and Endangered Species,. Henry Doorly Zoo, Omaha, NE, USA, 22 pages.

Graham, L.H., Byers, A.P., Armstrong, D.L., Loskutoff, N.M., Swanson, W.F., Wildt, D.E. and Brown, J.L. (2005) Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal endocrine analysis. General and Comparative Endocrinology 147:362-370.

Skidmore, J.A., Loskutoff, N.M and Billah, M. (2005) Developmental competence in vitro and in vivo of vitrified, hatched blastocysts from the dromedary camel (Camelus dromedarius). Reproduction, Fertility and Development 17:523-527.

Loskutoff, N.M., Huyser, C., Singh, R., Morfeld, K.A., Walker, D.L., Thornhill, A.R., Smith, M., Morris, L. and Webber, L. (2005) Removing HIV-1 and hepatitis C virus from semen: an improved method for washing sperm using a trypsinized silica particle suspension and a novel tube insert. Fertility and Sterility 84:1001-1010.

Huyser, C., Loskutoff, N.M., Singh, R. and Webber, L. (2005) O-212. Quantification of HIV-1 RNA in washed sperm samples. 21st Annual Meeting of the European Society of Human Reproduction and Embryology. Human Reproduction 20 (Suppl. 1): i79

Loskutoff, N.M., Huyser, C., Singh, R., Morfeld, K.A., Walker, D.L., Thornhill, A.R., Smith, M., Morris, L. and Webber, L. (2005) A novel and effective procedure for removing HIV and hepatitis B and C viruses from spiked human semen. Reproduction, Fertility and Development 17 (1,2): 185.

Morfeld, K.A., White, B., Mills, G., Krisher, R., Mellencamp, M.A. and Loskutoff, N.M. (2005) A novel sperm processing method for eliminating porcine reproductive and respiratory syndrome virus (PRRSv) from boar semen and its effects on embryo development in vitro and in vivo. Reproduction, Fertility and Development 17 (1,2): 186.

De la Rey, M., Morfeld, K.A., Treadwell, R. and Loskutoff, N.M. (2005) The effect of a novel semen disinfection treatment on the viability and fertilizing capacity in vivo of bovine spermatozoa. Reproduction, Fertility and Development 17 (1,2): 184.

Skidmore, J.A., Loskutoff, N.M and Billah, M. (2004) Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius). Reproduction, Fertility and Development 16:605-609.

Donoghue, A.M., Blore, P.J., Cole, K., Loskutoff, N.M. and Donoghue, D.J. (2004) Detection of Campylobacter or Salmonella in turkey semen and the ability of poultry semen extenders to reduce their concentrations. Poultry Science 83:1728-1733.

Loskutoff, N.M., Huyser, C., Singh, R., Morfeld, K.A., Walker, D., Thornhill, A.R., Smith, M., Morris, L., Webber, L. (2004) A novel and effective procedure for removing HIV-1 RNA from human semen. In: International Congress Series; Special Issue: Research Papers in Fertility and Reproductive Medicine. Proceedings of the 18th World Congress on Fertility and Sterility (IFFS 2004). Eds: Daya S., Pierson R. & Gunby J.; 1271C, pp. 200-204.

Huyser, C., Loskutoff, N.M., Singh, R. And Lindeque, K. (2004) A novel antibiotic cocktail for eliminating bacteria in human semen. In: International Congress Series; Special Issue: Research Papers in Fertility and Reproductive Medicine. Proceedings of the 18th World Congress on Fertility and Sterility (IFFS 2004). Eds:
Daya S., Pierson R. & Gunby J.; 1271C, pp. 205-209.

Loskutoff, N.M., Bowsher, T.R., Chatfield, J.A., Stones, G.A., Ramey, J.W., Zhang, L., Putman, M., Boland, C., Wharton, D. and Gardner, D.K. (2004) Ovarian stimulation, ultrasound-guided oocyte retrieval, ICSI and blastocyst production using sequential media in the western lowland gorilla (Gorilla gorilla gorilla). Reproduction, Fertility and Development 16:225.

Armstrong, D.L., Crichton, E.G., Dankof, S.M., Schwalbach, L.M.J., Gardner, D.K. and Loskutoff, N.M. (2004) Ovarian stimulation, laparoscopic oocyte retrieval, IVF and blastocyst production using sequential media in the African lion (Panthera leo). Reproduction, Fertility and Development 16:221.

Loskutoff, N.M., Singh, R., Huyser, C., Morfeld, K.A., Walker, D., Thornhill, A.R., Smith, M., Morris, L. and Webber, L. (2004) A novel and effective procedure for removing HIV from human semen. Proceedings of the International Federation of Fertility Societies, International Congress Series 1266, Montreal, Quebec, Canada
(20-25 May 2004).

Huyser, C., Loskutoff, N.M., Singh, R. And Lindeque, K. (2004) A novel antibiotic cocktail for eliminating bacteria in human semen. Proceedings of the International Federation of Fertility Societies, International Congress Series 1266, Montreal, Quebec, Canada (23-28 May 2004).

Loskutoff, N.M. (2003) Research programs
connecting African zoos and wildlife. Proceedings of the American Zoo and Aquarium Association (AZA) annual conference, Columbus, OH (7-11 September 2003).

Loskutoff, N.M. (2003) The South African Center for Conservation and Research. Proceedings of the African Zoo and Aquarium Association (PAAZAB) annual conference, Oudtshoorn, Klein Karoo, South Africa (3-6 June 2003)

Mastromonaco, G.F., Crawshaw, G., Loskutoff, N.M. and King, W.A. (2003) Cytogenetic screening of a family of gaur (Bos gaurus). Proceedings of the 13th North American Colloquium on Animal Cytogenetics and Gene Mapping, Louisville,
KY (13-17 July 2003).

Loskutoff, N.M., Morfeld, K. and Crichton, E.G. (2003) Trypsin activity after prolonged refrigerated storage. Theriogenology 59:384.

Crichton, E.G., Obringer, A., Lund, A., Kirshner, E., Pryor, W. and Loskutoff, N.M. (2003) Monitoring reproductive activity in the Tasmanian devil (Sarcophilus harrisii). Theriogenology 59:389.

Morfeld, K., Skidmore, J.A., Billah, A.M. and Loskutoff, N.M. (2003) Effect of various cryoprotectants and methods for the short- and long-term preservation of sperm from the dromedary camel (Camelus dromedarius). Theriogenology 59:400.

Kubisch, H.M., Rasmussen, T.A. and Loskutoff, N.M. (2003). Interferon-tau secretion by hybrid cattle x bison blastocysts and blastocyst outgrowths. Theriogenology 59:483.

Loskutoff, N.M. (2003) Role of embryo technologies in genetic management of conservation of wildlife. In: Reproductive Sciences and Integrated Conservation. W.V. Holt, A.R. Pickard, J.C. Rodger & D.E. Wildt (Eds.). Cambridge University Press, Cambridge, UK.

Loskutoff, N.M., Holt, W.V. and Bartels, P. (2003) Editors: Conservation Breeding Specialist Group (SSC/IUCN). Biomaterial Transport and Disease Risk: Workbook Development. CBSG, Apple Valley, MN.

Crichton, E.G., Bedows, E., Miller-Lindhom, A.K., Baldwin, D.M., Armstrong, D.L., Graham, L.H., Ford, J., Gjorret, J.O., Hyttel, P., Pope, C.E., Vajta, G. and Loskutoff, N.M. (2003) The efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biology of Reproduction 68: 105-113.

Gjorret, J.O., Crichton, E.G., Loskutoff, N.M., Armstrong, D.L. and Hyttel, P. (2002) Oocyte maturation, fertilization and early embryonic development in vitro in the Siberian tiger (Panthera tigris altaica). Molecular Reproduction and Development 63:79-88.

Loskutoff, N.M. (2002). Proceedings of the Second International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. Henry Doorly Zoo, Omaha, NE, USA, 322 pages.

Spindler, R.E., Gardner, D.K., Loskutoff, N.M., Critser, J. and Wildt, D.E. (2002) Beneficial impact of heterospecific companion embryo culture in the cat appears to be species dependent. Theriogenology 57:524.

Edens, M.S.D, Galik, P.K., Riddell, K.P., Givens, M.D., Stringfellow, D.A. and Loskutoff, N.M. (2002) Bovine herpesvirus-1 associated with single, trypsin-treated embryos is not infective for uterine tubal cells. Theriogenology 57:570.

Stander-Breedt, H., Schwalbach, L.M., Greylig, J. and Loskutoff, N.M. (2002). Effect of different cryodiluents and thawing methods on the motility and heterologous fertilizing capacity of African lion (Pathera leo) spermatozoa. Proceedings of the 9th International Symposium on Spermatology, Cape Town, South Africa (6-12 October 2002).

Motlomelo, K.C., Schwalbach, L.M., Greylig, J. and Loskutoff, N.M. (2002). A novel sperm cryopreservation method for the South African cheetah (Acinonyx jubatus). Proceedings of the 9th International Symposium on Spermatology, Cape Town, South Africa (6-12 October 2002).

Loskutoff, N.M. and Goodrowe, K. (2002). the AZA Reproductive Sciences Advisory Group and the IETS Parent Committee on Companion Animals, non-domestic and endangered species: novel resources for zoo and wildlife veterinarians. Proceedings of the American Association of Zoo Veterinarians annual conference, Milwaukee, WI.

Othen, L., Jarsky, T., French, J., Crichton, E., Loskutoff, N. and Bennett, C. (2002) Correlating ethological and physiological parameters as indicators of well-being in okapi (Okapia johnstoni) ACTH challenge, patterns of excretion, and impact of
introductions. Proceedings of the 2nd International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. Henry Doorly Zoo, Omaha, NE, USA., pp. 73-79.

O?Brien, J.K., Crichton, E.G., Evans, K.M., Schenk, J.L., Stojanov, T., Evans, G., Maxwell, W.M.C. and Loskutoff, N.M. (2002) Sex ratio modification using sperm sorting and assisted reproductive technology ? a population management strategy. Proceedings of the 2nd International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. Henry Doorly Zoo, Omaha, NE, USA., pp. 224-231.

Loskutoff, N.M. (2001) Giving nature a helping hand. American Zoo and Aquarium Association Communiqu?, February issue.

Bowsher, T.R. and Loskutoff, N.M. (2001) Assisted reproductive technologies in the great apes. Proceedings of the 1st International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. Henry
Doorly Zoo, Omaha, NE, USA, pp. 152-156.

Loskutoff, N.M. (2000) Assisted reproductive technology using sex-sorted sperm: an alternative strategy for the Gorilla SSP. Proceedings of the Bachelor Gorilla Workshop, Disney?s Animal Kingdom, Orlando, FL, USA.

Janesch, L., Rohr, J., Volenec, D., Grobler, D., Puffer, A., Prokupek, A.K., Armstrong, D.L., Dankoff, S., Curro, T., Simmons, H., Crichton, E.G., Hamilton, J., Rasmussen, L., Zimmermann, D., Lomneth, R., Wood, R., Wood, D. and Loskutoff, N.M. (2001) Post-thaw viability of wild cattle (Bos gaurus) and buffalo (Syncerus caffer) sperm cryopreserved using a novel, non-animal protein cryodiluent. Theriogenology 55:387.

Morfeld, K., Henton, M.M., Grobler, D., Bengis, R., Puffer, A., Armstrong, D.L., Dankoff, S., de la Rey, M., de la Rey, R., Hansen, H., Strick, J., Rasmussen, L., York, D., Zimmermann, D., de Klerk, L.M., van Dyk, D.S., Crichton, E.G., Popescu, A., Stringfellow, D. and Loskutoff, N.M. (2001). Elimination of Brucella abortus from infected domestic (Bos taurus) and wild (Bos gaurus) cattle and buffalo (Syncerus caffer) semen without compromising sperm viability. Theriogenology 55:393..

Puffer, A., de la Rey, R., de la Rey, M., Hansen, H., Grobler, D., Hofmeyr, M., Malan, J.H., Armstrong, D.L., Dankoff, S., Rasmussen, L., York, D., Zimmermann, D. and Loskutoff, N.M. (2001) Choice of chemicals used in immobilization protocols can significantly affect semen quality in ejaculates collected from free-ranging African buffalo (Syncerus caffer). Theriogenology 55:398.

Givens, M.D., Galik, P.K., Riddell, K.P., Stringfellow, D.A., Brock, K.V., Bishop, M.D., Eilertsen, K.J. and N.M. Loskutoff (2001) Reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells. Theriogenology 55:378.

Loskutoff, N.M. (2001). Proceedings of the First International Symposium on Assisted Reproductive Technology for the Conservation and Genetic Management of Wildlife. Henry Doorly Zoo, Omaha, NE, USA, 226 pages.

Hammer, C.J., Tyler, H.D., Loskutoff, N.M., Armstrong, D.L., Funk, D.J., Lindsey, B.K. and Simmons, L.G. (2001) Compromised development of calves (Bos gaurus) derived from in vitro-generated embryos and transferred interspecifically into domestic cattle (Bos taurus). Theriogenology 55:1447-1455.

Solti, L., Crichton, E.G., Loskutoff, N.M. and Cseh, S. (2000) Economical and ecological importance of indigenous livestock and the application of assisted reproduction to their preservation. Theriogenology 53:149-162.

Loskutoff, N.M. and Stroud, B. (2000) Editors: Proceedings of the 26th Annual Conference of the International Embryo Transfer Society. Theriogenology 53, No. 1 , pp. 1-535.

Loskutoff, N.M., Amstrong, D.L., Ohlrichs, C.L., Johnson, D.L., Funk, D.J., VanRoekel, P.V., Molina, J.A., Lindsey, B.R., Looney, C.R., Bellow, S.M., Hammer, C.J., Tyler, H.D. and Simmons, L.G. (2000) Transvaginal ultrasound-guided oocyte retrieval and the developmental competence of in vitro-produced embryo in vitro and in vivo in the gaur (Bos gaurus). Theriogenology 53:337.

Crichton, E.G., Armstrong, D.L., Vajta, G. and Loskutoff, N.M. (2000) Developmental competence in vitro of embryos produced from Siberian tigers (Panthera tigris altaica) cryopreserved by controlled rate freezing versus vitrification. Theriogenology 53: 328.

Morato, R.G., Crichton, E.G., Paz, R.C.R., Zuge, R.M., Moura, C.A., Nunes, A.L.V., Teixeira, R.H., Porto, L., Priscila, M.A.B.V., Guimaraes, S.H.R., Correa, S.H.R., Barnabe, R.C., Armstrong, D.L. and Loskutoff, N.M. (2000) Ovarian stimulation and successful in vitro fertilization in the jaguar (Panthera onca). Theriogenology 53:339.

Gjorret, J.O., Crichton, E.G., Armstrong, D.L., Loskutoff, N.M. and Hyttel, P. (2000) Oocyte maturation, fertilization and early embryonic development in vitro in the Siberian tiger (Panthera tigris altaica). Theriogenology 53:334.

Skidmore, J.A., Billah, M. and Loskutoff, N.M. (2000) Developmental competence of cryopreserved blastocysts collected from dromedary camels. Fertility 2000: Proceedings of the Joint Conference of the UK Fertility Societies, Edinburgh, UK.

Crichton, E.G., Armstrong, D.L., Graham, L.H., Bedows, E., Miller, A., Gjorret, J.O., Hyttel, P. and Loskutoff, N.M. (2000) The efficacy of porcine gonadotrophins for repeated stimulation of ovarian activity for in vitro embryo production in Siberian tigers (Panthera tigris altaica). Fertility 2000: Proceedings of the Joint Conference
of the UK Fertility Societies.

Finnegan, J.M., Loskutoff, N.M. and Brown, C.S. (1999) Cost effective method to Transport manually collected gorilla semen for long term storage. Proceedings of the American Association of Zoo Veterinarians, Columbus, Ohio.

Loskutoff, N.M. (1999) Interspecies embryo transfer ? a feasible strategy for safeguarding endangered species? Proceedings of the American Association of Zoo Veterinarians, Columbus, Ohio.

Loskutoff, N.M. (1999) How to preserve your own gametes at home (practical and generalized procedures for genome resource banking). Proceedings of the Association of Zoo Veterinary Technicians, Indianapolis, Indiana.

Loskutoff, N.M. (1999) The advantages and disadvantages of assisted reproductive technology (ART) for propagating exotic felids. Proceedings of the Pan African Association of Zoos and Botanical Gardens, Cape Town, South Africa.

Pope C, Schmid R, Mikota S, Dresser B, Pirie G, Lamb K, Godke R, Aguilar R, Loskutoff N. In vitro production of tiger (Panthera tigris) embryos after daily gonadotropin treatment and off-site laparoscopic oocyte retrieval. (1999) Proc. 7th Int. Conf. Breeding Endangered Species in Captivity, Cincinnati, Ohio, 1999; 244.

Skidmore, J.A. and Loskutoff, N.M. (1999) Developmental competence in vitro and in vivo of cryopreserved expanding blastocysts from the dromedary camel (Camelus dromedarius). Theriogenology 51:293.

Kurz, S.G., Healy, M.R., Loskutoff, N.M., Brown, C.S., Crichton, E.G., Barnes, A.M., Finnegan, J.M., Volenec, D. and DeJonge, C.J. (1999) In-vitro maturation and intracytoplasmic sperm injection of western lowland gorilla oocytes. Theriogenology 51:361.

Finnegan, J.M., Warnes, C.A., Belifore, M., Crichton, E.G., Volenec, D. and Loskutoff, N.M. (1999) Heterologous fertilizing capacity of cryopreserved sable antelope (Hippotragus niger) and scimitar-horned oryx (Oryx dammah) sperm using fresh and salt-stored bovine oocytes. Theriogenology 51:284.

Nelson, K.L., Crichton, E.G., Doty, L., Volenec, D., Finnegan, J.M., Morato, R.G., Pope, C.E., Dresser, B.L., Armstrong, D.L. and Loskutoff, N.M. (1999) Heterologous and homologous fertilizing capacity of cryopreserved felid sperm: a model for endangered species. Theriogenology 51:290.

Pope, C.E. and Loskutoff, N.M. (1999) Embryo transfer and semen technology from cattle applied to nondomestic artiodactylids. In: Zoo and Wild Animal Medicine: Current Therapy, 4th Edition. M.E. Fowler and R.E. Miller (Eds.), W.B. Saunders Co., Philadelpia pp. 597-604.

Loskutoff, N.M. (1998) Biology, technology and strategy of genetic resource banking in conservation programs for wildlife. In: Gametes: Development and Function, A. Lauria et al. (Eds.), Serono Symposia, Rome, Italy, pp. 275-286.

Brown, C.S. and Loskutoff, N.M. (1998) A training program for noninvasive semen collection in captive western lowland gorillas (Gorilla gorilla gorilla). Zoo Biology 17:143-151.

MacLean, R.A., Swanson, H., Hancock, J., O?Brien, E., Williams, K.R. and Loskutoff, N.M. (1998) Comparison of three techniques for separating motile sperm in cryopreserved Bos gaurus semen. Theriogenology 49:263.

Loskutoff, N.M. (1998) Assisted reproduction in western lowland gorillas. Proceedings
of the Pan African Association of Zoos and Botanical Gardens annual conference, Bloemfontein, Free State, South Africa.

Kurz, S.G., Barnes, A.M., Healy, M.R., Brown, C.S., Loskutoff, N.M., Armstrong, D.L., Ramey, J.W. and DeJonge, C.J. (1998) Comparative characteristics of semen collected by electroejaculation vs. Manual stimulation in the western lowland gorilla. Proceedings of the 8th International Congress on Spermatology, Montreal, Quebec, Canada.
O?Brien, E.S., Hoffman, S., Schawang, T., Suedmeyer, W.K., Easton, E., Schmitt, D. and Loskutoff, N.M. (1998) An update on studies to improve the short- and long-term storage of elephant semen. Proceedings of the 3rd International Elephant Research Symposium, Springfield, MO, USA.

Loskutoff, N.M. (1997) Okapi reproduction. Proceedings of the Okapi Metapopulation Workshop, White Oak Conservation Center, Yulee, FL, USA.

O?Brien, E., McCullough, K., Jefferies, E., Williams, K. and Loskutoff, N. (1997) Effect of different cryoprotectants on the short- and long-term storage of semen from the African elephant (Loxodonta africana). Proceedings of the 2nd International Elephant Research Symposium, Pittsburgh, PA, USA.

Watson R., Lanterna J., Garcia A., Cooper C., Tomsett G. and Loskutoff N.M. (1997) Freezing resistances of epididymal sperm from waterbuck, greater kudu and warthog using glycerol, ethanediol or DMSO. Theriogenology 47:411.

Rush E.M., Jewell M., Cooper C., Garcia A., Tomsett G. and Loskutoff N.M. (1997) Effect of cryoprotectant and thawing method for cryopreserving epididymal sperm from impala (Aepyceros melampus) for IVF. Theriogenology 47:406.

Schmid R., Lanterna J., Garcia A., Tomsett G. and Loskutoff N.M. (1997) A comparison of standard bovine and equine cryodiluents for cryopreserving epididymal sperm from greater kudu (Tragelaphus strepsiceros). Theriogenology 47:407.

Winger Q., Damiani P. and Loskutoff N.M. (1997) The application of standard bovine protocols for the maturation and fertilization of blesbok (Damaliscus dorcas phillipsi) oocytes using epididymal sperm cryopreserved in DMSO or glycerol. Theriogenology 47:412.

Pope, C.E., Dresser B.L., Chin N.W., Liu J.H, Loskutoff N.M., Behnke E.J., Brown C., McRae M.A., Sinoway C.E., Campbell M.K., Cameron K.N., Owens O.M., Johnson C.A., Evans R.R. and Cedars M.I. (1997). Birth of a western lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer. American Journal of Primatology 41:247-260.

Meintjes, M., Bezuidenhout, C., Bartels, P., Visser, D.S., Meintjes, J., Loskutoff, N.M., Le, R., Fourie, F., Barry, D.M. and Godke, R.A. (1997). In vitro fertilization of in vitro matured oocytes recovered from free-ranging Burchell's (Equus burchelli) and Hartmann's (Equus zebra hartmannae) zebra. Journal of Zoo & Wildlife Medicine 28:251-259.

Loskutoff, N.M., Simmons, H.A., Goulding, M., Thompson, G., De Jong, T. and Simmons, L.G. (1996) Species and individual variations in cryoprotectant toxicities and freezing resistances of epididymal sperm from African antelope. Animal Reproduction Science 42:527-535.

Kurz, S.G., Barnes, A.M., Ramey, J.W., Brown, C., Loskutoff, N.M., Simmons, L.G., Armstrong, D.L. and De Jonge, C.J. (1996) Semen characteristics of a western lowland gorilla determined by manual and computer-assisted motion analysis. Biology of Reproduction 54, Suppl. 1: 301.

Dresser, B., Pope, E., Chin, N., Liu, J., Loskutoff, N.M., Behnke, E., Brown, C., McRae, M., Sinoway, C., Campbell, M., Cameron, K., Evans, R., Owens, O., Johnson, C. and Cedars, M. (1996) Successful in vitro fertilization, embryo transfer and pregnancy in a western lowland gorilla. Theriogenology 45:248.

Loskutoff N.M. (1996) Reproductive biotechnology for the conservation and genetic management of wildlife. Embryo Transfer Newsletter, International Embryo Transfer Society Vol. 14, No. 4, pp. 23-26.

Buice, R., Maloy, D., Raath, C. and Loskutoff, N.M. (1995) Effects of various cryoprotectants on epididymal sperm from the African elephant (Loxodonta africana). Proceedings of the Association of Zoo Veterinary Technicians, Baltimore, Maryland.

Armstrong, D.L., Looney, C.R., Lindsey, B.R., Gonseth, C.L., Johnson, D.L., Williams, K.R., Simmons, L.G. and Loskutoff, N.M. (1995) Transvaginal egg retrieval and in-vitro embryo production in gaur (Bos gaurus) with establishment of interspecies pregnancy. Theriogenology 43:162.

Shaw, D.G., Kidson, A., van Schalkwyk, J.O., Bartels, P., Loskutoff, N.M., Bezuidenhout, C., Barry D.M. and Lishman, A.W. (1995) In-vitro production of African buffalo (Syncerus caffer) embryos derived from follicular oocytes and epididymal sperm. Theriogenology 43:322.

Kidson, A., Loskutoff, N.M., Raath, C., Wood, C.A., Williams, K.R., van Schalkwyk, J.O., Dyche, W.K., Barry, D.M. and Bartels, P. (1995) Age- and parity- dependent differences in ovarian activity and oocyte maturity in the African elephant (Loxodonta africana). Theriogenology 43:246.

Meintjes, M., Bartels P., Bezuidenhout, C., Visser, D.S., Meintjes, J., Loskutoff, N.M., Fourie, F. le R., Barry, D.M. and Godke, R.A. (1995) In-vitro fertilization of in-vitro matured oocytes recovered from free-ranging zebra in South Africa. Theriogenology 43:279.

Fordyce-Boyer, R., Sanger, T., Loskutoff, N.M., Kumamoto, A., Johnston, L. and Armstrong, D. (1995) Comparative cytogenetic study of the roan and sable antelope, Hippotragus equinus and Hippotragus niger. Applied Cytogenetics 21:189-191.

Loskutoff, N.M., Bartels, P., Meintjes, M., Godke, R.A. and Schiewe, M.C. (1995) Assisted reproductive technologies in nondomestic ungulates: a model approach to preserving and managing genetic diversity. Theriogenology 43:3-12.

Schiewe, M.C., Loskutoff, N.M., Durrant, B.S., Johnston, L.A., Armstrong, D.L. and Simmons, L.G. (1994) Gaur sperm cryopreservation trial: analysis of packaging type and rapid freezing method for potential field application. Theriogenology 41:291.

Loskutoff, N.M. and Bartels, P. (1994) Assisted reproductive technology for the preservation and management of genetic diversity in wildlife species. Proceedings of the 11th LatinAmerican Congress on Genetics, Puerto Vallarta, Mexico, 25-30 September 1994.

Staff

Dr. Jason Herrick,
Director of Reproductive Sciences

Jonathan T. Aaltonen, M.H.S.
Reproductive Sciences Lab Supervisor