What Is An Induce Ovulator? Induced Ovulator Animals

Abstract

Induced ovulation maximizes captive convenance success, increasing productivity and facilitating the contribution of otherwise infertile animals to the gene pool. In marsupials, induced ovulation to produce fertile young is unknown. Here we present an induction protocol efficient in inducing not-cycling and not-reproductive females to bike, mate, ovulate, and conceive. Ovulation was induced in Sminthopsis macroura using an initial injection of 0.06 IU equine serum gonadotropin (eSG)/g (time 0), followed on day 4 past 0.04 IU eSG/g. Using this induction regime, the timing of follicular and embryonic evolution mimics natural cycles and results in the nascency of viable, fertile young. Response to induction is not significantly affected past animal historic period, making this protocol an constructive conservation tool. We have established a time-table of evolution post-obit induction, providing a source of precisely timed inquiry material. This is the first induced ovulation protocol in any marsupial to result in demonstrated fertile offspring and to allow the reliable collection of known-historic period samples during both the follicular stage and the gestation menses.

Introduction

Induced ovulation maximizes captive breeding success, assuasive increased productivity of domestic species, laboratory colonies, and the development of more effective convenance programs for endangered species. Although the reproductive patterns of many marsupials are well characterized (Tyndale-Biscoe & Renfree 1987), knowledge of the hormonal changes associated with mating, ovulation, and fertilization remains scarce (Hinds et al. 1996). As a consequence, the development of induced ovulation protocols for marsupial species has relied largely on proven eutherian treatments and application in marsupials has had varying success (Hinds et al. 1996).

The marsupial ovary is highly sensitive to handling with eutherian gonadotropins (Rodger et al. 1992a, Hinds et al. 1996). Responses differ betwixt species, and successful awarding requires species-specific test and refinement. In full general, the optimum dose required to induce ovulation in marsupials appears much lower than reported in eutherians. Numerous authors have reported ovarian abnormalities in marsupials following over stimulation with exogenous hormones (Harding 1969, Renfree et al. 1988, Rodger & Mate 1988, Molinia et al. 1998, Hickford et al. 2001, Menkhorst et al. 2007). Over stimulation, associated with an elevated concentration of plasma estradiol (Renfree et al. 1988), may result in degenerative ovarian changes such as cysts, premature follicle luteinization, and oocyte retention (Rodger 1990).

Among the studied monovular marsupials, induced ovulation has been described in Macropus eugenii (Renfree et al. 1988, Magarey et al. 2003), Trichosurus vulpecula (Harding 1969, Rodger & Mate 1988, Glazier & Molinia 1998, 2002, Glazier 1999), and Bettongia penicillata (Hayman et al. 1990, Rodger et al. 1992b). Induced ovulation protocols have as well been applied to polyovular marsupials including, Monodelphis domestica (Nelson & White 1941), Dasyuroides byrnei (Fletcher 1983), Sminthopsis crassicaudata (Smith & Godfrey 1970, Rodger et al. 1992a, Hinds et al. 1996, Mate 1998), and Sminthopsis macroura (Hickford et al. 2001, Menkhorst et al. 2007); still, an induction protocol that can result in normal embryos is rare, and a protocol that tin can result in the birth of normal and fertile pouch immature (PY) from an induced ovulation cycle of previously not-cycling or not reproductive females has yet to be successfully established.

South. macroura is a polyestrous, polyovular, seasonally convenance dasyurid (Woolley 1990), constitute in arid and semi-arid regions of northern and central Australia. Information technology has an estrous bike length of 23.25 days (Woolley 1990) and the shortest mean gestation period (10.7 days) of any known mammal (Selwood & Woolley 1991). In some cases where development of the corpora lutea occurs at a faster rate, the gestation menstruation can exist as short as 9.5 days (Selwood & Woolley 1991). In natural cycles, the get-go of the follicular phase of the estrous bike (time 0) occurs 24 h earlier the first day of weight rise and/or the advent of cornified epithelial cells in the urine. Estrus corresponds with days 4–vi of the follicular phase, and spontaneous ovulation occurs on day 7 (Selwood & Woolley 1991). Intensive reproductive monitoring of this species in our long-term colony has allowed authentic determination of estrous cycle phase, twenty-four hour period of ovulation, gestation period, and stage of pregnancy, providing an animate being model in which the timing of hormonal administration can be accurately controlled.

Successful induction has been demonstrated in S. macroura using a diversity of different induction regimes and hormonal concentrations (Hickford et al. 2001, Menkhorst et al. 2007). In investigating appropriate dosages, time of dosage commitment, time of ovulation and oviducal transit time, Hickford et al. (2001) and Menkhorst et al. (2007) have established that in that location is no pregnant deviation in induction results between non-circadian, intermediate, follicular, and gestation phases of the estrous cycle, excluding the part of the luteal stage where progesterone concentration is high (Hickford et al. 2001, Menkhorst et al. 2007). Typically, equine serum gonadotropin (eSG), administered every bit two injections (Hickford et al. 2001, Menkhorst et al. 2007), successfully induced ovulation of more oocytes per ovary than did ovulation in animals undergoing natural cycles and if mated, conceptuses resulted. Nonetheless, both Hickford et al. (2001) and Menkhorst et al. (2007) noted an increase in the occurrence of ovarian abnormalities, a scenario preferably avoided for the breeding of endangered animals. Following induction, the frequency of atretic or prematurely luteinized ovarian follicles appeared greater in the study by Menkhorst et al. (2007), who employed a hormone dose comparatively larger than Hickford et al. (2001). Neither of the previous S. macroura induced ovulation studies resulted in the birth of PY, although previous not-cycling females began to cycle naturally following consecration and one gave birth to a litter in her first natural cycle post-obit induced ovulation (Menkhorst et al. 2007). Rodger et al. (1992a) induced South. crassicaudata to produce PY, only the reproductive condition of the female person was unclear and the study did not demonstrate whether the offspring produced were fertile.

The aims of the electric current study were: i) to found an induction protocol in South. macroura that was like shooting fish in a barrel to utilize and led to the provision of a time-table of follicular and embryonic development to enable the drove of precisely timed inquiry material and ii) to determine the fertility of offspring produced following induced ovulation.

Results

The 57 females induced to ovulate in this study savage into 3 reproductive categories, 26 were cycling and reproductive, 20 were cycling but non-reproductive, and 11 were non-cycling. Despite deliberately selecting animals with poor reproductive functioning, 97% (55/57) of induced females responded to the consecration protocol, with simply two non-responders (one cycling and reproductive in its beginning convenance season; ane non-cycling in its 2d breeding season). Of the animals examined later on twenty-four hour period 6 post injection, ninety% (43/48) showed evidence of successful ovulation. The five animals that did not ovulate include the two not-responders and 3 animals in their first convenance season (ii cycling and reproductive; one cycling merely non-reproductive) that contained atretic follicles inside their ovaries. Creature age had no pregnant effect on the ability of animals to begin cycling (t ₍₅₅₎=0.80, P=0.45) and/or ovulate (t ₍₄₆₎=0.21, P=0.84) post-obit induction.

Follicle development

Ovarian follicle development was examined in nine induced animals during the 2nd half of the follicular phase (days 4–6.vii post initial injection) and compared with normal evolution in naturally cycling animals, north=29 (Fig. 1). Post-obit induction, developing follicles showed normal morphology, and follicle diameter measurements recorded were within the range expected for naturally cycling animals (Kress et al. 2001, Nation & Selwood 2009). Primordial, principal, and early secondary follicles were nowadays in all examined ovaries. By twenty-four hour period 4 postal service injection (due north=iii), ovaries independent late tertiary stage follicles, with antral pocketing evident among cells of the granulosa layer. Antral stage follicles, containing antral lacunae of varying size and germinal vesicle stage oocytes were found between days 5 and 6.vii mail injection (n=6). Oocytes from the largest and most advanced antral follicles (examined half-dozen.7 days post-injection) remained in meiotic abort, with centrally full-bodied cytoplasmic vesicles, and adherent cumulus cells. The most mature oocytes showed developing cytoplasmic polarity and the beginnings of perivitelline space formation; however, they had withal to undergo germinal vesicle breakdown, extrude the first polar body, and shed the cumulus layer, essential events which forestall ovulation.

In comparison to previous studies (Hickford et al. 2001, Menkhorst et al. 2007), the incidence of follicular abnormalities (premature follicular luteinization, follicular atrophy, and retained oocytes) was low in this report, although still significantly college than in the ovaries of naturally cycling animals (Table 1).

Table 1

Summary of the proportion of induced and naturally cycling Sminthopsis macroura used for the assay of follicular, embryonic, and parthenogenetic development, pouch young (PY) production, and failed experiments; and a comparison of the number of corpora lutea or antral follicles and abnormal follicles betwixt induced and naturally cycling animals.

		Reproductive status (n)
Stage and type of follicles and conceptuses	Animals (n)	NC	CNR	CR	Mean±due south.e.thousand. corpora lutea or antral follicles per ovary	Mean±s.e.m. abnormal follicles per ovary
Induced (due north=57)
Follicles	nine	2	four	3	12.47±0.61^a (due north=43)	i.77±0.29^b (n=43)
Embryos	19	2	seven	x
Parthenotes	15	3	3	9
Pouch immature	2	1	0	1
Failed
Responded and mated just no PY	1	0	ane	0
Responded but did non mate	6	2	4	0
Failed
Atretic	three	0	i	2
Non-responsive	2	i	0	1
Total		xi	20	26
Natural (n=73)
Follicles	29	All cycling and reproductive			10.36±0.28^a (north=73)	0.55±0.eleven^b (n=73)
Embryos	29
Parthenotes	15
					^a(t=3.xvi, P=0.002)	^b(t=3.94, P<0.0001)

Female reproductive status is represented as: NC, not-cycling; CNR, cycling just non-reproductive; CR, cycling and reproductive. Values with different superscripts differ significantly; P<0.05.

Comparison of the number of developing antral follicles or corpora lutea in the ovaries of induced and naturally cycling animals revealed that the induction protocol significantly increased the number of oocytes selected for maturation and subsequent ovulation (Table 1).

Embryonic development

A full of 19 induced animals were used for the timely collection of conceptuses in order to compare the rate of embryonic development with conceptuses obtained from natural cycles (n=29). Of these, ten were cycling and reproductive, 7 were cycling only non-reproductive, and two were non-cycling.

Using induced ovulation, fertilized oocytes were detected in oviducts as early as 167 h (or at 7.0 days) after assistants of the first injection. Uterine zygotes were detected at 7.2 days, 2-prison cell conceptuses at vii.6 days, 4-cell conceptuses at 7.eight days, 16-jail cell conceptuses at 8.6 days, and unilaminar blastocysts at 10.ix days (Fig. 1). Bilaminar blastocysts were detected at 7.25 days post ovulation (Fig. one). All conceptuses were of normal morphology. Since ovulation occurs seven days mail first injection, when these analyses were compared with the timely collection of conceptuses from naturally cycling animals, there is no significant difference in the timing of ovulation and embryonic development (Fig. 1). Nosotros accept collected embryos from two induced and three naturally cycling females that appeared to develop faster than normal, probably having the shorter gestation period of 9.5 days shown by Selwood & Woolley (1991).

Parthenote development

Post-obit consecration, parthenotes were collected from the uteri of 15 non-mated females (n=15) during the start three days of gestation and the rate of parthenogenetic evolution compared with the samples retrieved from naturally cycling animals (n=15). In general, parthenotes appeared to develop faster following induction. During the first 24-h post-ovulation (solar day i of gestation), the uteri of naturally cycling animals yielded merely unfertilized oocytes, whereas 2- and iv-cell parthenotes were collected from induced animals. On day 2 of gestation, naturally cycling animals yielded parthenotes with a maximum of four cells, compared with 8-prison cell parthenotes found in the uteri of induced animals. Past twenty-four hours 3 of gestation, both the induced and naturally cycling animals independent 8-jail cell phase parthenotes (maximum cell number), and no further parthenogenetic development was observed.

Pouch young

An additional nine animals (one cycling and reproductive, five cycling but non-reproductive, and three not-cycling) were induced for the nascency of offspring (PY). Following consecration, 3 females mated successfully (determined past the presence of sperm in urine samples) and 2 of these gave birth to feasible young. A litter of six PY was born to a previously not-cycling female at xvi.7 days post initial injection, corresponding to a gestation period of 10–ten.3 days. Another female person, cycling and reproductive, gave birth to a litter of half-dozen PY at 17.7 days post outset injection, corresponding to a gestation menstruation of eleven–11.three days. PY from both litters survived, grew to normal adult size and weight, and were fertile in the following breeding flavor. The male offspring mated successfully, and the female offspring gave birth to feasible young, producing 4 or five PY per litter.

Discussion

In this study, all not-cycling females bar i responded to the induction protocol and began to cycle. All not-cycling females, examined after day six post injection, had ovulated. This highlights the success of the current protocol in recruiting previously non-cycling females to cycle and ovulate. In addition, all cycling merely non-reproductive females responded to the hormone government, with just one failing to ovulate.

Following induction, fertilized tubal oocytes were detected in oviducts 7.0 days after the beginning injection, congruent with the end of the 7-day follicular phase and the day of ovulation in naturally cycling animals. In this study, induced animals ovulated early in the morning time, consequent with the findings of Menkhorst et al. (2007).

Prior to ovulation, the developing oocytes of advanced antral follicles must resume meiosis and undergo a serial of maturational events essential for fertilization (Merry et al. 1995). Our findings indicate that the transition of immature germinal vesicle stage oocytes to mature metaphase II stage oocytes may be an extremely rapid process, with meiotic maturation, ovulation, and oviducal fertilization all occurring within a period of <8 h.

Embryonic evolution of conceptuses obtained post-obit induced ovulation appears to mimic the timing of embryonic development in naturally cycling animals. From time 0, minimum collection times were vii.0 days for tubal oocytes and seven.2 days for uterine zygotes, making oviducal transit possibly every bit short as 4.viii h. Length of the gestational period following induction, 10–11 days, was within the range expected for this species (Selwood & Woolley 1991). Embryos from a small number of animals appeared to be developing faster than normal, all the same, this is not unusual as the gestation menstruation can be as short every bit nine.five days (Selwood & Woolley 1991) in naturally cycling animals. This may be due to a faster embryonic evolution at the end of the breeding flavor, possibly a mechanism to maximize reproductive success.

Induced ovulation is a routine laboratory procedure in mice, pioneered by Runner & Gates (1954), and has been successfully applied to other small laboratory species such as rats (Goh et al. 1992, Jiang et al. 1999) and rabbits (Treloar et al. 1997), equally well as larger domestic eutherians such as moo-cow (Donaldson & Ward 1986, Lopes da Costa et al. 2001), horse (Niswender et al. 2003), sheep (Leoni et al. 2001), pig (Amirov et al. 1998), and caprine animal (Kiessling et al. 1986). In mice, induced ovulation is used non but for the production of large numbers of oocytes, but also for the establishment of timed pregnancies that proceed to term with the birth of young (Edwards & Gates 1959, Edwards & Fowler 1960, Beaumont & Smith 1975, Spindle & Goldstein 1975). Despite this, protocols in other mammals have focussed largely on the ability to obtain greater numbers of oocytes or embryos for research purposes and often ignored its potential awarding for conservation purposes.

Induced ovulation may exist used to increase the productivity of captive populations, enhancing the reproductive potential of aged, non-cycling or previously non-reproductive females and regulating the timing of mating, ovulation, and fertilization. The protocol presented here, in which hormone dosages are calculated co-ordinate to torso weight, should be easily adjusted for application in related dasyurid species. Within Commonwealth of australia, 17/64 extant dasyurid taxa are threatened (Maxwell et al. 1996); however, only one species, Parantechinus apicalis, has been systematically bred in captivity for release (Moro 2002). By establishing an induced ovulation protocol effective in the product of live and fertile litters, we are ane step closer to aiding the recovery of endangered species through captive breeding coupled with reintroduction or translocation. In the present study, animals responded to consecration regardless of age, making this protocol an even more than effective conservation tool as age is unknown in many zoo animals collected from the wild.

We present here for the first time, an induced ovulation protocol that is 90% effective in inducing both the cycling and not-cycling females to ovulate, with reduced occurrence of follicle atresia or luteinization, results in normal embryonic development provided that mating occurred successfully and ultimately the production of fertile young. The viability of embryos resulting from induced ovulation was confirmed by taking pregnancies to total term with a fully established fourth dimension-table of follicular and embryonic evolution. This time-table can be utilized to enable the collection of precisely timed research material, increasing productivity and reducing loss. The successful production of viable, fertile immature, following induced ovulation has enormous potential in increasing the productivity of captive marsupial colonies and assisting the conservation of endangered species through enhanced breeding programs.

Materials and Methods

Animals

The stripe-faced dunnarts used in this study were from a laboratory colony maintained at the University of Melbourne, Department of Zoology, following Australian National Health and Medical Enquiry Council Guidelines for the Intendance and Use of Animals for Scientific Purposes and held under permits issued by the Department of Sustainability and Surroundings.

Reproductive monitoring

The reproductive status of all the females was adamant by daily monitoring of weight, cells in urine samples, and pouch changes throughout the breeding season (Hickford et al. 2001). The follicular phase of the estrous period is associated with a transient increase then autumn in weight over 7 days, and the presence of cornified epithelial cells in the urine. If conceptuses are required, a male person is introduced when cornified epithelial cells peak, usually betwixt days 4 and six of the follicular phase, and mating is detected by the presence of spermatozoa in the urine. Spontaneous ovulation (time 0 of gestation) occurs at the stop of the follicular stage and is associated with a fall in weight and the advent of many polymorphonuclear leucocytes in the urine. The post-obit day, marked by an increase in weight, is the get-go day of gestation. The luteal phase is contained inside the gestation phase and is maintained until day 9 of gestation. Southward. macroura has a mean gestational catamenia of 10.7 days (Selwood & Woolley 1991). The stop of gestation is indicated past a precipitous drop in weight, accompanied by xanthous crystals and sometimes crimson blood cells in the urine, a clear secretion in the pouch and PY if pregnant (Selwood & Cui 2006). The flow between the stop of these changes in a non-meaning cycle and the beginning of the next follicular phase is termed the intermediate phase.

Treatments

The majority of animals employed in this study were in their beginning breeding season; 85% (62/73) of control (naturally cycling) and 84% (48/57) of experimental (induced to ovulate). The remaining animals were used during their second breeding season. Females were grouped according to their reproductive status: cycling and reproductive, cycling but not-reproductive, and non-cycling. Cycling females that had either failed to mate or had mated only produced no PY over one–eight months of reproductive monitoring were categorized every bit cycling but non-reproductive. In this study, all control animals were cycling and reproductive, whereas many animals selected for induction showed poor reproductive operation. Cycling females, whether reproductive or non-reproductive, were induced during the follicular, late luteal (twenty-four hour period 6–x following ovulation), or intermediate stage of the estrous cycle, equally treatment during early-mid luteal phase has proven less successful (Menkhorst et al. 2007). Females in which the estrous cycle could not exist adamant by daily monitoring (not-cycling females) were induced to stimulate their showtime cycle and/or regulate their estrous cycle contour. Ovulation was induced using a series of 2 hormone injections delivered in calcium- and magnesium-free PBS. Menkhorst et al. (2007) establish no difference in the time of ovulation subsequently morning time or evening stimulation, so to make hormone administration more convenient, we gave both the injections at 1600 h and also returned to the employ of body weight to summate the advisable hormone dosage, giving each female a total of 0.ane IU eSG/g. At time 0, animals received a dose of 0.06 IU eSG/thou (Folligon; Allhank Trading Co., S Melbourne, Victoria, Australia), followed on day 4 by a 2d injection of 0.04 IU eSG/k (Hickford et al. 2001). The book required per injection was determined for individual females on the 24-hour interval of induction, and hand-warmed prior to i.p. injection.

Analysis

Females were killed by inhalation of Halothane (Rhone Merieux, Westward Footscray, Victoria, Australia), followed past cervical dislocation. The reproductive tracts were removed, washed in warmed PBS⁻ (35 °C) and examined nether a dissecting microscope (Zeiss, Due north Ryde, New South Wales, Australia) to confirm estrous cycle phase. An inverted microscope (Wild Leitz, Melbourne, Victoria, Australia) with heated stage was employed for closer examination of ovarian follicles, oocytes, zygotes, conceptuses, and parthenotes. To examine follicle development, ovaries were collected on the final iii days of the follicular stage of the estrous cycle. Mean follicle diameters were calculated past measuring the longest axis, and the axis perpendicular, using a calibrated ocular micrometer. Follicles were determined to exist principal, secondary, third, or antral based on size and morphology (Kress et al. 2001, Nation & Selwood 2009). Antral follicles were carefully ruptured using 29 G needles to appraise oocyte maturity (Merry et al. 1995). As ovulation occurs at 0700 h (Menkhorst et al. 2007), females were killed belatedly on the day of ovulation and on subsequent days (at closer intervals during days 1–3 of gestation) to collect phase-specific conceptuses. Parthenotes were collected from not-mated females during the commencement three days of the gestational catamenia to confirm ovulation and to examine the rate of parthenogenetic development. Ovarian corpora lutea were counted to decide ovulation number and corpora albicans to constitute previous cycling action. Oviducts were examined under a transmitted light microscope for the presence of oocytes. Each uterus was transferred to warmed culture medium (DMEM; Sigma–Aldrich), slit longitudinally forth the midline and carefully inverted so embryos or parthenotes, if present, could ringlet out gently into the medium. Embryos were examined for bear witness of fertilization, and assessed for developmental phase and normality (Selwood & McCallum 1987). PY born following induced ovulation were raised in the colony and their fertility was assessed during the post-obit breeding flavor by incorporating them into the normal breeding programme.

Statistical assay

Independent sample t-tests were employed to examine the event of animal age on the power of females to begin cycling and/or ovulate following consecration. The number of developing antral follicles, corpora lutea and atretic follicles per ovary was calculated every bit the mean (±s.e.grand.) and compared between induced and naturally cycling groups by independent sample t-tests. Values were considered statistically significant when P<0.05.

Declaration of interest

The authors declare that in that location is no conflict of interest that would prejudice the impartiality of this scientific work.

Funding

This piece of work was supported by the University of Melbourne and by the Foundation for Research Science and Engineering, New Zealand (Grant No. MELB0301).

Acknowledgements

We would like to give thanks Mrs Kamani Indrika Nanayakkara, Mr Hsien Chun Aloysius Ng and Ms Heidi Snow for their contribution to animal maintenance.

References

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