Successful artificial insemination in the koala (Phascolarctos cinereus) using extended and extended-chilled semen collected by electroejaculation

Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.     

Semen collection and evaluation in free‐ranging Brazilian rattlesnakes (Crotalus durissus terrificus)

Two hundred‐ninety species of reptiles are estimated to need urgent action for conservation, with at least 113 threatened species worldwide. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species includes 80 species of snakes, with six native Brazilian species, a number likely to be an underestimation. Some authors believe that assisted reproduction would be an important tool to improve reproduction in captivity of some reptiles. An efficient technique for semen collection and evaluation is an important step in development of protocols for cryopreservation of semen or artificial insemination in snakes, contributing to the conservation of endangered species. Although these techniques are important, some basic semen parameters are described for four of the ～2,900 snake species in the world. The Brazilian rattlesnake (Crotalus durissus terrificus) was chosen as a model for semen collection in snakes because it is found quite often in Sao Paulo State. Semen was collected once from each animal by the same investigator during the mating season of this species in Brazil. After antiseptic cleansing of the skin around the cloaca, the snakes were injected subcutaneously with a dose of 15 mg/kg of 1% solution of lidocaine around the cloaca. Semen was collected with ventral massages after cloacal relaxation and directly from genital papilla inside the cloaca. A total of 28 ejaculates from 39 animals were obtained, representing collection efficiency of 71.80%. Semen volume and concentration in Brazilian rattlesnakes ranged from 3–70 µl and from 0.94–2.23 × 10⁹ spermatozoa/ml, respectively. Zoo Biol 0:1–6, 2007. © 2007 Wiley‐Liss, Inc.     

First successful artificial insemination with frozen-thawed semen in rhinoceros

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of ∼135 × 10⁶ motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of ∼500 × 10⁶ motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.     

Spermophagy in semen in the red wolf,Canis rufus

The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley-Liss, Inc.     

Ultrasound examination and behavior scoring of captive broadnose sevengill sharks,Notorynchus cepedianus (Peron, 1807)

Serial ultrasound examination of four mature female sevengill sharks (Notorynchus cepedianus) was carried out over 18 months. Monitoring the reproductive cycle and development of follicles and fetuses in sharks in a noninvasive manner using this technique has not been reported previously. Sharks were caught out of the “Oceanarium” tank by divers using a specially made catch‐out bag, and brought to a holding area for examination. A behavior scoring system was used to monitor the impact of regular handling on the well‐being of the animals. Ultrasound showed the growth and regression of follicles in sevengill ovaries, and allowed an approximation of the reproductive stage of these sharks. Monitoring behavior at five time points during the procedure showed that regular handling of sharks for clinical studies could be done with minimal impact on animal welfare. The ability to follow reproductive events in elasmobranches using ultrasonography is an important step in the application of assisted reproductive technology in these species. Assisted reproductive technology, such as monitoring female reproductive cycles and artificial insemination, could potentially be used to maintain genetic diversity and compliment aquaria‐based breeding programs for endangered species such as the gray nurse shark (Carcharias taurus). Zoo Biol 26:383–395, 2007. © 2007 Wiley‐Liss, Inc.     

Collection of semen by manual stimulation and ejaculate characteristics of the black flying‐fox (Pteropus alecto)

Semen collection and preservation is the first step toward the development of an artificial insemination program in endangered Pteropus spp. Semen was collected by manual stimulation from a single “human‐habituated” P. alecto. Manual stimulation resulted in the successful collection of motile spermatozoa on 17 of 34 attempts. The semen had a pH of 8.2 (n=2). With the exception of volume, seminal characteristics (concentration, motility, acrosome and plasma membrane status) were similar to those collected previously by electro‐ejaculation. Zoo Biol 27:159–164, 2008. © 2008 Wiley‐Liss, Inc.     

Semen characteristics of the quaker parakeet (Myiopsitta monachus)

This report describes characteristics of semen samples collected from eight captive quaker parakeets (Myiopsitta monachus) using the massage technique. Overall, semen characteristics were (mean±SE, range in parentheses, n=8 males): ejaculate volume 1.96±0.26μl (1.02–2.96), sperm concentration 346.6±64.6 million/ml (74.8–579.3), and number of sperm per ejaculate 0.71±0.16 million (0.09–1.53). Significant differences were observed between males for all three semen characteristics. Semen pH for the eight males ranged from 8.05 to 8.5. The semen samples were collected early in the breeding season, so the data reported may not be representative of ejaculates from males in peak breeding condition. However, this study provides the first rigorous semen data from this species and demonstrates that good‐quality semen samples, suitable for artificial insemination, can be collected regularly from quaker parakeets using the massage technique. Zoo Biol 21:507–512, 2002. © 2002 Wiley‐Liss, Inc.     

北京动物园大熊猫繁殖记录

北京动物园在三十年中,共繁殖大熊猫28胎（其中双胎18次）,产仔46只,成活21只,成活率为45．65％,为大熊猫的迁地保护作出了杰出的贡献。1963年第一次在人工饲养下自然繁殖成功大熊猫“明明”;1978年第一次用人工授精方法繁殖成功大熊猫“元晶”;1980年第一次以纯超低温保存的大熊猫冷冻精液人工授精成功产下二仔;1987年第一次在人工饲养下繁殖出子三代;1990年第一次实现不同地理位置的大熊猫自然交配繁殖成功幼仔“亚庆”;1992年第一次繁殖成功大熊猫人工授精第三代幼仔“京京”;1992年第一次以人工授精繁殖长大的雄性大熊猫自然交配成功繁殖“永明”和“永亮”;1992～1993年第一次以人工哺育的方法使未食母乳的大熊猫幼仔“永亮”成活;1993年第一次达到1992年所产的3仔全部成活,成活率为100％。     

Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranus after one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranusafter one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Assessment of motility,acrosomal integrity,and viability of giant panda (Ailuropoda melanoleuca) sperm following short‐term storage at 4°C

Short‐term cool storage of semen affords many of the same benefits as cryopreservation, without the extensive cryoinjury to sperm associated with freezing. Semen storage for artificial insemination (AI) is an integral part of giant panda captive breeding programs. AI functions to generate offspring from males that have never bred, are underrepresented, or are unable to breed with genetically desirable females due to geographic separation. In the present study, semen was collected by electroejaculation from six giant pandas and extended in four media (TEST with 0% or 5% glycerol, or SFS with 0% or 5% glycerol). Subsequently, initial motility (MOT), speed of progression (SOP), percent live (% L), and percent normal acrosome (% NAR) values were recorded. These parameters were assessed again at 4, 8, 24, and 48 hr of incubation at 4°C in each medium. Motility scores (MS) were calculated as MOT×SOP². The MS of each sample was also recorded after the addition of 20 mM caffeine. Results were expressed as a percent of the initial value (% I). Although all three parameters decreased over time, the MS decreased at a faster rate than either the % L or the % NAR. There were significant differences between individual pandas for each measured parameter, while only % IMS was significantly affected by medium (P=.0006). The addition of caffeine increased % IMS at all time periods and reduced the differences between media to nonsignificant levels. The addition of glycerol was not beneficial for short‐term semen storage. These data indicate that storage of giant panda semen at 4°C for up to 48 hr maintains sperm viability at a level sufficient for use in an AI program. Zoo Biol 22:529–544, 2003. © 2003 Wiley‐Liss, Inc.     

Cryopreservation of houbara semen: A pilot study

The potential of producing viable houbara bustard Chlamydotis undulata undulata progeny with cryopreserved semen was investigated during the 1998 breeding season. Houbara semen was diluted and rapidly frozen into pellet form in LN₂ using dimethylacetamide (DMA) as a cryoprotectant. Thawed semen exhibited 24.86± 10.61% recovery of fresh sperm motility levels and 36.44±16.17% of fresh viability counts. Two second‐year females were inseminated on two and three occasions, respectively, with thawed semen. One female laid four eggs between two clutches and the second female laid two eggs in a single clutch. All eggs were fertile; two developed to 18–21days but failed to pip; one chick died at pip and three chicks hatched. This is the first documented occasion that houbara bustard chicks were conceived with cryopreserved semen and it demonstrates the feasibility of using a rapid, simple, and relatively inexpensive methodology to achieve this result. Zoo Biol 18:147–152, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Efficient embryonic culture method for the Japanese striped snake,Elaphe quadrivirgata,and its early developmental stages

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named “sausage‐style (SS) culture”, allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113–115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full‐color images with high‐resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.     

Semen preservation and artificial insemination in domesticated South American camelids

Semen preservation and artificial insemination in South American camelids are reviewed giving emphasis to work done in Peru and by the authors. Reports on semen evaluation and the preservation process indicate that semen of alpacas and llamas can be manipulated by making it liquid first. Collagenase appears to be the best enzyme to eliminate viscosity. Tris buffer solution maintains a higher motility than egg-yolk citrate, phosphate buffered saline (PBS), Triladyl, and Merck-I extenders. Cooling of semen took 1 h after collected, and equilibrated with 7% glycerol presented a better motility and spermatozoa survival at 1, 7, 15 and 30 days after being slowly frozen in 0.25 mL plastic straws. Trials of artificial insemination with freshly diluted semen and frozen–thawed semen are encouraging and needs to be tested extensively under field conditions. Recently, fertility rates varied from 3 to 67%. Semen preservation and most important, artificial insemination appear to be a reality, and could be used to improve the genetic quality of alpacas and llamas.     

Somatic polyploidy in extraembryonic membranes of the snake,Elaphe guttata

Summary Video-densitometric DNA measurements of Feulgenstained tissues of 42 day old eggs of the corn snake,Elaphe g. guttata (Columbridae, Serpentes), revealed a basic DNA content of 2C=2.17 pg, with somatic polyploidy in the allantois, the chorioallontois, the yolk sac, and other extraembryonic membranes. The maximum value determined was 128C (in binucleate cells 2×128C) at the distal pole of the egg. This is the first report of somatic polyploidy in a snake, and one of the first in reptiles in general.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.     

Artificial insemination of Reindeer (Ragifer tarandus)

Semen collected from reindeer bulls in an artificial vagina was used for the artificial insemination of 16 reindeer cows: two with undiluted semen, five with diluted semen and nine with frozen semen. The two cows which received undiluted semen gave birth to normal calves 217 days later. The other cows returned to oestrus 23.5 days (mean) after insemination and subsequently calved, having been served by a fertile bull.     

Noninvasive semen collection from an adult orangutan

Preservation of the genetic diversity of the captive orangutan, especially the wild-caught founders, is critical in maintaining a long-term population in zoological parks. One solution to the problem of maintaining maximum genetic diversity would be to initiate a program of artificial insemination for genetically underrepresented individuals through the banking and interinstitutional use of cryopreserved semen. However, little is known about basic orangutan semen characteristics, and current methodology is inadequate to support such a program. In this paper, we report the results of semen collection from an adult Sumatran orangutan (Pongo pygmaeus abelli), using an artificial vagina without anesthesia or electrical stimulation. A total of 27 ejaculates were evaluated during a 1-year period. The total and liquid volumes of the ejaculates at 1 h following collection were 6.1 ± 0.6 ml and 2.6 ± 0.4 ml, respectively (mean ± SEM). The liquid portion continued to exude semen for 2 h; however, 90% of the motile sperm was exuded within the first 30 min. The total number of sperm in the ejaculate was 164 ± 10⁶ ± 16.5, and the percentage of motile cells was 60 ± 2.7%. We conclude that the artificial vagina provides a promising technique for semen collection in the orangutan, and view these results as an initial step in developing methods for in vitro sperm capacitation, sperm cryopreservation, and artificial insemination. © 1993 Wiley-Liss, Inc.     

Captive breeding and reintroduction of the oriental pied hornbill (Anthracoceros albirostris) in Khao Kheow Open Zoo,Thailand

This article discusses two related issues of the captive breeding and reintroduction of the Oriental pied hornbill (Anthracoceros albirostris) in order to increase its population in the natural habitats. Oriental pied hornbills were bred in Khao Kheow Open Zoo. Three pairs were separated and kept in breeding cages. Females occupied artificial nests between February and April 2005–2007. Eggs were laid and incubated between February and March each year from 2005 to 2007. Nestlings hatched in late March and left the nest in late April 2005–2007. Each breeding pair was fed with approximately 400 g of food each day. All three pairs reproduced resulting in mature offspring of seven in 2005, six in 2006, and five in 2007. Four of sixteen 3‐year‐old birds were randomly selected and equipped with a GPS receiver on their backs. Activities of the birds attached and unattached with GPS were not significantly different. The first two birds (one female, one male with GPS) were reintroduced on August 5, 2006, and another nine birds (four birds with GPS) were reintroduced on December 26, 2006. The average home range of these reintroduced birds was 0.13 km². Their foods consisted of wild plants and animals in the home range. The first reintroduced pair was able to breed naturally by laying and hatching eggs in an artificial nest. Two juveniles left the nest in April 2008. These results indicate that both captive breeding and reintroduction are potentially important ways to increase the population of the Oriental pied hornbill in natural habitats. Zoo Biol 31:683‐693, 2012. © 2011 Wiley Periodicals, Inc.