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Reproductive Technology; |
Schools of Animal Studies,3 and Land, Crop and Food Sciences,4 The University of Queensland, Gatton, Queensland 4343, Australia
Dreamworld,5 Coomera, Queensland 4209, Australia
Currumbin Wildlife Sanctuary,6 Currumbin, Queensland 4223, Australia
Environmental Protection Agency,7 Brisbane, Queensland 4000, Australia
Institute of Zoology,8 London NW1 4RY, England
Centre for Mined Land Rehabilitation9 and School of Biomedical Sciences,10 The University of Queensland, St. Lucia, Queensland 4072, Australia
ABSTRACT
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.
induced ovulator, LH surge, luteal phase, marsupial, pouch young
Artificial insemination (AI) has the potential to be very useful in the reproductive and genetic management of zoo or fragmented wild populations. The utility of the technique is significantly increased if it is incorporated with the use of frozen-thawed spermatozoa and genome resource banking. Although Australia has many endangered marsupials that are currently managed in zoological institutions or by state and federal environmental agencies, the use of assisted breeding in these populations is presently confined to a small number of research projects.
One limitation to the use of assisted breeding in marsupials is the relatively scant knowledge of the reproductive physiology that controls the reproductive cycle [1]. One exception to this rule has been the development of assisted breeding technology in the koala which, at the commencement of this study, had resulted in the successful production of 13 offspring [2, 3]. The only other successful example of AI in a marsupial that has produced a pouch young (PY) is that of the tammar wallaby (Macropus eugenii) [4]. All of the koalas born by AI to date have been produced with semen collected by an artificial vagina (AV), and there has been no reported attempt to use electroejaculated (EE) or diluted semen.
A major advantage of using AI is that it should be possible to extend semen with diluents in order to inseminate several females using the same ejaculate. Semen must also be extended for successful short-term storage or for cryopreservation. This potentially presents a problem for koala AI, as recent studies have indicated that koala semen may contain an ovulation-inducing factor, as has been described for camels, alpacas, and llamas [5–8]. It is possible that extension of koala semen might result in a reduction of the ovulatory capacity of the semen, thus potentially requiring artificial induction of ovulation with exogenous hormones.
Presently, the quality of cryopreserved koala semen after thawing is inadequate for successful AI [9]. Although this precludes genome resource banking for the moment, the international and national transport of koala semen for management of zoo populations only requires semen to be stored for the duration of shipment, which for current applications should take no longer than 72 h. Previous studies of extended-chilled koala semen have shown that spermatozoa are capable of maintaining motility for upwards of 42 days at 5°C [10], but the fertilizing potential of these spermatozoa was not evaluated.
Another important component in the success of an AI program is the timing of insemination. The koala has an estrus of approximately 10 days [11], but earlier studies involved the use of females in the first half of their behavioral estrus, and the success of ovulation induction through natural mating or via AI in the latter stages of estrus has not been evaluated. If normal gestation ensues following insemination in the second half of estrus, then this would allow a sufficient time frame for the logistical arrangements associated with the national or international shipment of extended-chilled semen.
The aims of this study were to investigate whether AI with extended and/or extended-chilled semen could induce a luteal phase and subsequent production of PY. A further aim was to determine whether the female koala remains fertile throughout the entire 10 days over which behavioral estrus is observed.
This study was conducted in association with two wildlife parks in the Gold Coast region of Australia (Currumbin Wildlife Sanctuary and Dreamworld) over three breeding seasons (September to March), beginning in spring 2004 and ending in early autumn 2007. All 42 female and 28 male sexually mature koalas were kept under standard husbandry conditions as outlined by Blanshard [12] and Jackson et al. [13]. All research animals remained clinically healthy throughout the study period, with male and female koalas always being housed in separate enclosures. Decisions on which males were to be used as semen donors were primarily dictated by the available sires and the breeding plan of each wildlife park. The details of animal use for each experiment are described below. This study was conducted under the approval of The University of Queensland Animal Ethics Committee (SAS/659/05/SAS/KEGE/ARC and SAS/426/06/ARC).
Anesthesia and Semen Collection
Electroejaculation of koalas was conducted under general gaseous anesthesia using a protocol modified from McGowan et al. [14]. When a satisfactory level of anesthesia was achieved, a 4.5- to 5-mm diameter endotracheal tube (Tyco Healthcare, Kendall Curity, Thailand) was guided into the trachea using a rigid endoscope. The EE procedure in koalas has been described in detail by Johnston et al. [15, 16]. Electroejaculation was conducted without incident or trauma over the duration of the study.
Following semen collection, sperm concentration of the original semen sample was estimated using a Makler sperm counting chamber (Sefi-Medical Instruments, Haifa, Israel). A semen sample (10 µl) was diluted (1:10) in a Tris-citrate glucose buffer (TCG; 0.25 M Tris base, 0.08 M citric acid, and 0.07 M glucose) [10] and placed on a prewarmed (35°C; normal koala body temperature) microscope slide with a coverslip for the evaluation of percentage of motility and rate of forward progression (using a subjective scale from 1–5 defined by Barth, 1995 [17]) under a phase-contrast microscope (400x magnification) with warm stage set at 35°C.
The AI procedure used in this study has been described in detail by Johnston et al. [2, 3]. Briefly, once a female had been identified in estrus [11], she was brought into the veterinary surgery and held in a "4-pinned" full restraint position (Fig. 1). Initially, the cloaca was exposed and a glass stimulator instrument, custom made with protuberances to mimic the spines of the koala glans penis, was inserted into the urogenital sinus [3]. The stimulator, which was lightly lubricated (K-Y Lubricating Jelly; Johnson & Johnson), was moved back and forth approximately 40 times over a 20-sec period (Fig. 1A) [3, 18]. Previous studies have shown that using the glass stimulator prior to insemination results in a higher rate of ovulation and luteal phase induction [3]. The AI catheter (model SP VIBC1270; SurgiVet) was then inserted inside the urogenital sinus, and the cuff of the catheter was gently inflated with approximately 1 ml saline to secure it in place (Fig. 1B). Semen was delivered into the catheter by a 1-ml syringe (Terumo; Terumo Corp.), and semen deposition in the urogenital sinus was optimized by discharging the remaining semen in the catheter with 1 ml air. The catheter was then held in place in the restrained animal for approximately 5 min.
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Venipuncture and Hormonal Analysis
To characterize and monitor ovulation and subsequent pregnancy following AI or natural mating, a 1-ml blood sample was taken from the female immediately after insemination or mating (T0) and then 24, 28, and 32 h after AI or mating to analyze the plasma for a LH surge. Additionally, blood samples were collected on Days 14 and 28 after AI or mating in order to detect a rise in plasma progestogen concentration indicative of induction of a luteal phase. Koala venipuncture and blood sample processing procedures were undertaken as previously documented [11].
Plasma samples were assayed for LH concentration using a heterologous RIA with antiserum GDN15 and ovine LH as standard and radiogland [19]. This assay system has previously been validated for koala plasma [3]. The interassay coefficients of variation were 14.3% (n = 4; mean = 2.3 ng/ml), 15.0% (n = 7; mean = 6.9 ng/ml), and 5.9% (n = 5; mean = 11.2 ng/ml). The lowest detection limit of the assay was 0.4 ng/ml, and the highest was 25.0 ng/ml. Samples above the detection limit of the assay were diluted with LH-free sheep plasma to determine the concentration of the sample. All samples were assayed in duplicate.
Plasma samples were also assayed for progestogen concentration using Spectria Progesterone RIA kits (Orion Diagnostica). The assay was validated for koala plasma by demonstrating parallelism between dilutions of pooled plasma and the standard curve (F1,8 = 3.89; P = 0.089). The intraassay coefficient of variation was 8.3% (n = 8; mean = 7.7 ng/ml), and interassay coefficients of variation were 15.4% (n = 20; mean = 2.9 ng/ml) and 15.8% (n = 21; mean = 7.8 ng/ml), respectively. A recovery of 71.2% was obtained when progesterone was added to a plasma sample before assay and the values reported were not corrected for these losses. The lowest detection limit of the assay was 0.1 ng/ml, and the highest was 31.4 ng/ml. Samples above the detection limit of the assay were diluted with progesterone-free gelding plasma to determine the concentration of the sample. All samples were assayed in duplicate. The progesterone antibody cross-reacted with progesterone 100%, pregnenolone 3.9%, and <1% with all other steroids tested (Spectria specifications).
Confirmation of Induction of a Luteal Phase
The most obvious way of measuring ovulatory success in the koala is by the birth of PY at around 34–36 days after AI. However, if the female fails to give birth there are three other indirect means to determine reproductive status. The first technique involves the length of time taken for the female to come back into estrus. Johnston et al. [11] have determined that a long estrous cycle of approximately 50 days is typically a result of the successful formation of a corpus luteum, whereas a failure to ovulate and enter a luteal phase is associated with a short estrous cycle of approximately 30 days. Both of these outcomes can also be validated by measurements in the concentration of plasma LH and progestogen at key points following insemination. Assessment of behavioral estrus was conducted by "teasing" the females every day. This involved introducing a male koala (under close supervision) into the enclosure and observing the female's response. This is a well-established method of estrus detection in this species [11, 12]. Induction of a luteal phase was confirmed by the presence of a plasma LH surge within 24–32 h after insemination [3] and also by an elevated plasma progestogen concentration on Day 14 and/or 28 after AI [11].
Experiment 1: AI Using Undiluted and Extended Semen Collected by EE
This experiment was conducted to investigate whether dilution of koala semen has an effect on the ovulation induction capacity of the semen and to ensure that EE semen is suitable for AI. In this experiment, 23 different female and 13 different male koalas were used. Artificial insemination was conducted with semen allocated into three treatments with nine females in each. In treatment 1, females received 1 ml undiluted semen that was collected by EE. The volume of semen chosen was based on the amount of ejaculate previously determined from collecting semen using an AV [20]. In treatment 2, 2 ml semen collected by EE that had been previously extended 1:1 with a Tris-citrate buffer and antibiotics [21] was deposited into each female. The volume of 2 ml semen was chosen because even though the semen was diluted, it would still contain the same total sperm number and amount of putative ovulation induction factor as in treatment 1. Treatment 3 used only 1 ml diluted semen for insemination in order to determine whether dilution of sperm numbers and any ovulation induction factor would have a detrimental effect on the successful ovulation and production of PY. Blood samples were collected after insemination to determine the relative concentrations of plasma LH (0, 24, 28, and 32 h) and progestogen (0, 14, and 28 days).
Experiment 2: AI With Extended-Chilled Semen Collected by EE
This experiment was conducted to investigate whether it was possible to produce koala PY following AI with extended-chilled (24 and 72 h) semen. In this experiment, 26 different female and 14 different male koalas were used. Artificial insemination was conducted with semen samples that were manipulated into three treatments; there were nine females per treatment. Treatment 1 of this study was the same as treatment 2 in experiment 1, in which females received 2 ml diluted semen within 15 min of its collection. In treatment 2, 2 ml diluted semen was placed into the female after chilling the spermatozoa for 24 h. Treatment 3 was the same as treatment 2, but semen was chilled for 72 h. Extended semen was placed into a 5-ml glass test tube (Borex; Chase Scientific, Rockwood, TN) and chilled slowly by placing it directly into a commercially available chilled semen transport container (Equitainer; Hamilton Research, Inc.) which, according to the manufacturer, reaches the final internal temperature of 5°C within 10 h and provides refrigeration for up to 3 days. To gain a more accurate estimate of the temperature within the Equitainer, an "i-button" temperature logger (DS1922L; Maxim Integrated Products Inc.) was also placed inside the shipping container adjacent to the semen sample. This device recorded the internal temperature of the container every 10 min. Upon insemination, the semen was not deliberately rewarmed to 35°C, but inseminated in a chilled state and allowed to rewarm slowly in the reproductive tract of the female koala. An estimate of percentage of motility and rate of sperm movement was conducted directly after the collection of koala semen (but prior to extension and chilling), as well as before AI of the chilled semen. Blood samples were collected after insemination to determine the relative concentrations of plasma LH (0, 24, 28, and 32 h) and progestogen (0, 14, and 28 days)
Experiment 3: Natural Mating of Koalas in the Latter Stages of Behavioral Estrus
This experiment was conducted to determine whether it was possible to induce ovulation to produce fertilized koala oocytes and the production of PY from females in the latter stages of their behavioral estrus. In this experiment, 18 different female and 17 different male koalas were used. Females were naturally mated 8 or 10 days following their first expression of behavioral estrus. Blood samples were collected after insemination to determine the relative concentrations of plasma LH (0, 24, 28, and 32 h) and progestogen (0, 14, and 28 days).
The effects of the treatments on semen quality parameters were assessed using ANOVA, allowing for the effects of males used multiple times. Changes in semen quality associated with storage in experiment 2 were assessed using paired t-tests. Treatment effects on AI outcome were evaluated using an exact 
2 test. Changes in plasma concentrations of LH and progestogen following AI and natural mating were analyzed using a repeated-measures ANOVA with an ante-dependence error structure [22]. Hormone concentrations were log transformed prior to analysis, and in addition the results were confirmed with a Kruskal-Wallis nonparametric test [23]. All analysis was carried out using the SAS statistical system, version 8.2 2001.
The mean ± SEM (range) total number of spermatozoa inseminated in treatments 1, 2, and 3 was 44.3 ± 16.3 (6–150) x 106, 30.3 ± 7.6 (12–82) x 106, and 20.1 ± 8.5 (3–75) x 106, respectively. The mean ± SEM (range) motility of semen samples used was 77.7% ± 4.3% (50%–91%) in treatment 1, 75.9% ± 3.1% (63%–89%) in treatment 2, and 74.7% ± 3.2% (62%–88%) in treatment 3. The mean ± SEM (range) rate of sperm movement for treatment 1 was 3.7 ± 0.3 (3–5), 3.7 ± 0.2 (3–4.5) for treatment 2, and 3.4 ± 0.3 (2–4.5) for treatment 3. There were no statistical differences in any of the seminal characteristics measured between each treatment.
There was no statistical difference in mean plasma LH concentrations between each treatment, nor was there any difference between the mean ± SEM maximum LH concentrations of 13.5 ± 3.5 ng/ml, 14.8 ± 3.5 ng/ml, and 11.9 ± 4.0 ng/ml obtained for treatments 1, 2, and 3, respectively. All three treatments were successful in inducing ovulation and in producing PY. Luteal phase induction occurred in seven of nine females, with four of the seven females giving birth in both treatments 1 and 2. In treatment 3, six females had a luteal phase, and four of these six subsequently had PY.
The lowest total number of spermatozoa in one ejaculate that was inseminated and resulted in the successful production of PY was 3 million. The overall mean ± SEM total number of spermatozoa inseminated that resulted in the successful production of offspring was 26.9 ± 6.1 million. The mean ± SEM (range) of progressive motility and rate of sperm movement that resulted in the production of offspring was 76.3% ± 1.8% (67%–85%) and 3.8 ± 0.2 (3–5), respectively.
The percentage of progressive motility (mean ± SEM; range) of koala spermatozoa before (78.6% ± 3.5%; 65%–92%) and after chilling for 24 h (79.7% ± 3.4%; 57%–88%) was not significantly different. The percentage of progressive motility (mean ± SEM; range) of koala spermatozoa before (76.3% ± 4.2%; 58%–90%) and after (71.2% ± 2.5%; 60%–84%) chilling for 72 h was also not significantly different. Figure 2 shows the mean temperature recorded by the i-button within the Equitainer (n = 18). The mean ± SEM temperatures at 0, 24, and 72 h of refrigeration in the Equitainer were 27.5°C ± 0.8°C, 10.0°C ± 0.7°C, and 25.0°C ± 0.2°C, respectively.
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There was no statistical difference in mean plasma LH concentrations between each treatment at 24, 28, and 32 h following AI. All three treatments were successful in inducing ovulation. Treatments 1 and 2 induced a luteal phase in five of nine females, whereas treatment 3 produced a luteal phase in six of the nine females. Although treatment 1 failed to result in the production of any PY, semen chilled for either 24 h or 72 h and then placed into the female resulted in the birth of two and four PY from nine AI attempts, respectively.
The overall mean ± SEM total sperm number inseminated that resulted in the successful production of offspring was 38.2 ± 12.1 million. The lowest sperm number in an inseminate to result in the successful production of PY was 7 million. The mean ± SEM (range) of progressive motility and rate of motility prior to insemination that resulted in the production of offspring was 73.0% ± 2.2% (65%–78%) and 4.1 ± 0.4 (3–5), respectively.
There was no statistical difference in mean plasma LH concentrations between each treatment at 24, 28, and 32 h following mating. Of the 12 females naturally mated on Day 8 of oestrus, 50% gave birth (6 of 12); only 30% of 10 females that were naturally mated on Day 10 produced PY. All of the females in this experiment had a confirmed luteal phase, except for one female that was mated on Day 10.
General Observations on Periovulatory LH Secretion
Table 1 shows the mean (± SEM) plasma LH concentrations (24, 28, and 32 h and maximum LH concentrations) following AI or natural mating of females in experiments 1–3. The data have been pooled for anovulatory cycles, cycles incorporating a nonpregnant luteal phase, and pregnant females, regardless of experiment or treatment, to allow comparison between these outcomes. The results presented in Table 1 indicate that pregnant females had a significantly (P < 0.05) higher plasma LH concentration 28 and 32 h after AI or natural mating than those females that had a luteal phase but failed to give birth. Plasma progestogen concentrations at Days 14 and 28 were not significantly different between pregnant and nonpregnant females.
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The offspring produced by AI in experiment 1 are the first marsupial PY born following the use of extended semen collected by EE, an achievement that provides an additional and highly effective approach for management of koala reproduction and genetics. Previous koala AI success had been based on the use of semen collected by an AV, which greatly restricts the availability of potential donors. But now that it has been demonstrated that semen collected by EE can be used to produce PY, it will be possible to use semen from free-ranging males and from males in zoological institutions that have not been trained to service an AV. Potentially, semen from wild koalas can now be collected in the field, extended, and transported for use in captive populations or transferred into fragmented populations that have suffered a serious loss of genetic diversity.
The conception rate achieved in experiment 1 was 44%; this compares favorably with estimates of conception rates following natural mating in established zoological institutions that specialize in koalas (e.g., Lone Pine Koala Sanctuary) of between 43% and 57% [24]. The 44% conception rate reported in this study also is similar to that determined previously by Johnston et al. [2] when using a combination of semen collected with an AV together with natural and artificial methods of ovulation induction. The koalas used in this study were not selected on the basis of particularly favorable reproductive history, but on a breeding plan to maximize genetic diversity within the captive colonies.
A principal hypothesis tested in experiment 1 was that dilution of koala semen would have an effect on the ovulation induction capacity of the inseminate. The results of this study indicated that AI of undiluted and extended semen resulted in no significant difference with respect to the successful induction of either a luteal phase or the production of offspring. This finding indicates that koala semen maintains its ovulation inducing capacity when extended to a 1:1 dilution.
One of the most interesting findings in experiment 1 was the successful production of PY following insemination with only 3 million spermatozoa. A similar finding was also observed in experiment 2, where a PY was born following the insemination of only 7 million spermatozoa that had been chilled for 72 h. This suggests that sperm numbers can be quite low but still achieve conception when using AI techniques in the koala.
The offspring produced by AI in experiment 2 are the first marsupial PY to be born following the use of extended-chilled semen. This achievement opens the way for the national and international exchange of koala genetic material. A period of 72 h is sufficient time to allow for international air transportation to relevant overseas zoological institutions currently housing koalas. There is also the possibility that koala semen could be stored chilled for periods longer than 72 h, as Johnston et al. [10] have observed that motility can be maintained for upwards of 8 days at 5°C without any significant loss of motility.
An unexpected result in experiment 2 was the inability of the commercial semen shipping container to reach and maintain semen at the desired temperature (5°C), although this did not appear to have an observable detrimental effect on semen quality. Nevertheless, this demonstrates the value of using a temperature data logger within the shipment device for transport over long periods.
One of the chilled semen samples that was used in the successful production of a PY was an ejaculate that had been collected in one wildlife park and then inseminated into a female in another sanctuary approximately 43 km away. This represents a significant development in the use of an artificial breeding technology in a marsupial for the purposes of genetic exchange and reproductive management between institutions.
One possible explanation for the lack of AI success of treatment 1 of experiment 2 was that by chance, for some reason the randomly selected animals used in this treatment were not as reproductively sound as those used in experiment 1 (which was conducted approximately 1 yr previously). However, this explanation appears unlikely, as four of the seven females used in experiment 2 produced PY in experiment 1. Similarly, in treatment 2 of experiment 2, three of the seven koalas that did not give birth had previously produced PY a year before in experiment 1. It should be noted that all the females that had PY the previous breeding season had sufficiently recovered their body condition postweaning before being used for AI in experiment 2. It is also unlikely that semen quality might account for the lack of AI success in treatment 1 of experiment 2, as the semen quality (percentage of motility, rate of sperm movement, and sperm concentration) was similar across all three treatments.
Koalas have a behavioral estrus that lasts on average 10 days [11]. Typically, previous studies of natural breeding or AI have mated or inseminated koalas on Days 2–5 of estrus [2, 3, 11, 18] in the belief that mating koalas later in estrus could lead to reduced ovulation rates, ovulation of poor-quality oocytes, or an inability of the follicle to convert into a functional corpus luteum. The ability to mate and successfully produce PY in a female koala later in her estrus has the advantage of extending the time frame within which AI can be conducted. For example, if a koala is detected in estrus on Day 1 and is capable of ovulating a fertile oocyte 7–8 days later, this would provide ample time in which to prepare for the collection and disease screening of semen prior transporting it for AI. Experiment 3 demonstrates that natural mating of koalas on Day 8 of estrus results in a conception rate of 50% (6 of 12), a result which is similar to previous estimates of conception success of natural matings, which usually occurred earlier in estrus [24]. It remains to be seen whether the same rate of conception can be achieved with AI. Interestingly, when female koalas were naturally mated on Day 10, the conception rate fell to approximately 30%. This may be associated with some form of follicular atresia and/or ovulation of an aged oocyte or an inability of the follicular cells to luteinize.
When LH data from all three experiments were combined, irrespective of whether the insemination was artificial or natural, a total of 19 anovulatory cycles, 24 nonpregnant luteal phases, and 20 pregnancies were observed. No marsupial studies have produced a comparable dataset, and therefore some additional analysis was warranted. These data have provided a quantitative reassessment of the timing of the LH surge in the koala and a better understanding of the threshold LH concentration that might be differentially associated with induction of luteal phase and successful pregnancy. In pregnant koalas (but not in the ovulatory nonpregnant cycles), the highest mean concentration of plasma LH occurred 28 h after insemination. Clearly, a LH surge is required in order to induce a luteal phase and/or pregnancy, and it is interesting that the plasma LH concentration in pregnant animals was significantly (P < 0.05) higher than that found in koalas that showed a nonpregnant luteal phase. Perhaps there is a threshold level of LH surge secretion in the koala that is required to induce ovulation and/or adequate luteal function and therefore maintain pregnancy. However, evaluation of luteal function is only based on two observations of plasma progestogen concentrations (Days 14 and 28) following AI or natural mating, and it is therefore insufficient to conclude that luteal function is not different between pregnant and nonpregnant.
If koala semen does possess an ovulation-inducing factor in its seminal plasma [3], as is the case in camelid semen [5–8], then this may account for some of the variability in the success of ovulation and induction of a luteal phase. Electroejaculation is likely to produce an ejaculate of varying seminal biochemical composition, depending on the relative dimensions of the reproductive tract and the skill of the operator. Such variability in seminal composition could produce variability in the ovulation induction capacity of the semen. Consequently, future AI protocols based on EE semen may need to be combined with other artificial methods of ovulation induction, such as the use of GnRH, LH, or human chorionic gonadotropin. Further studies are required to explore the ovulation induction capacity of koala semen, comparable to those conducted in camelid species [5–8].
The theoretical and practical significance of this study resides in its demonstration that it is now possible to use extended and extended-chilled koala semen collected by EE for AI to produce koala offspring. It has also demonstrated that koalas can be successfully mated up to Day 8 of estrus and produce PY. These findings provide crucial information for the further development of koala reproductive and genetic management based on the use of assisted breeding. In total, 31 koalas have now been produced using AI, making the koala program one of the most successful assisted breeding programs for wildlife species in the world. The next steps in the development of this technology are the production of koalas by using frozen-thawed spermatozoa for AI and the establishment of a functional genome resource bank for the species.
ACKNOWLEDGMENTS
We are grateful for the assistance of veterinary staff, zookeepers, and volunteers at Dreamworld and Currumbin Wildlife Sanctuary for their dedicated efforts in the detection of oestrus and blood sample collection. We also thank Ms. Lyn Knott (School of Veterinary Science) and Ms. Tamara Keeley (Western Plains Zoo) for their advice regarding the validation procedures for the progesterone assay.
FOOTNOTES
1Supported by an Australian Research Council (ARC) Linkage Grant and the Koala Enhanced Genetic Exchange Program (KEGEP). 
2Correspondence and current address: Camryn D. Allen, 20947 North 55th Ave., Glendale, AZ 85308. FAX: 623 215 8599; e-mail: c.allen1{at}uq.edu.au
Received: 2 August 2007.
First decision: 26 August 2007.
Accepted: 10 December 2007.
REFERENCES
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