In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9802-x
Meiotic maturation of oocytes recovered from the ovaries of Indian big cats at postmortem Brahmasani Sambasiva Rao & Yelisetti Uma Mahesh & Komjeti Suman & Katari Venu Charan & Rhisita Nath & K Ramachander Rao
Received: 25 March 2014 / Accepted: 22 July 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract In order to increase the available sources of genetic material for endangered members of the great cat family, this study was designed to assess the meiotic competence of oocytes recovered from postmortem ovaries of the Indian leopard, tiger and lion. The average number of oocytes that were recovered per ovary was 11.0±5.0, 11.0±3.5 and 21.3± 8.8 for tiger, lion and leopard, respectively. The proportion of culture grade oocytes for tiger, lion and leopard were 72.7, 78.8 and 71.9%, respectively. The culture grade oocytes were matured in tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 μg/ml FSH, 6 IU/ml LH, 1 μg/ml 17β-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. After in vitro maturation, 56.3, 53.8 and 58.7% of the tiger, lion and leopard oocytes, respectively, were matured. The proportion of oocytes that extruded first polar body was significantly higher when the oocytes were collected from the animals of less than 15 yr of age compared to above 15 yr. These findings suggest that the oocytes recovered from ovaries of tiger, lion and leopard immediately postmortem can be successfully matured to MII stage.
Keywords In vitro maturation . Oocyte . Leopard . Tiger . Lion
B. S. Rao (*) : Y. U. Mahesh : K. Suman : K. V. Charan : R. Nath : K. R. Rao Laboratory for Conservation of the Endangered Species (LaCONES), CSIR—Centre for Cellular and Molecular Biology (CSIR-CCMB), Uppal Road, Hyderabad 500 007, India e-mail: [email protected]
Introduction The cat family (Felidae) is represented by 37 species of which all members except the domestic cat are classified as threatened, vulnerable or endangered (Nowell and Jackson 1996). The Asiatic lion (Panthera leo persica), Indian tiger (Panthera tigris tigris) and Indian leopard (Panthera pardus fusca) are considered as Schedule I (endangered) animals by the Indian Wild Life Protection Act (1972) due to their decline in population in alarming numbers. The reasons attributed for their decline are habitat loss, depletion of prey base, competition from other predators and poaching, and range decline is considered a strong indicator of population decline (Dinerstein et al. 2007). The International Union for Conservation of Nature (IUCN) has classified the Asiatic lion and the Indian tiger as endangered and the Indian leopard as near threatened (IUCN 2014). In order to increase the population size of the wild/endangered species, apart from management practices, application of assisted reproductive technologies (ART) would be necessary for the success of captive breeding programmes (Wildt et al. 1986; Pope 2000; Holt et al. 2004; Swanson 2006; Andrabi and Maxwell 2007). However, the application of ART for the propagation of wild/endangered species is limited by several factors (Pukazhenthi et al. 2006; Roldan et al. 2006), such as the limited number of animals and lack of availability of species-specific biological material required for a better understanding of the fundamental biology of gametes (Wildt et al. 1986). It is in this context that recovery of gametes from wild/endangered animals which have died due to accidents, medical reasons, disease outbreaks and old age could serve as a resource, to understand the fundamental physiology of the species concerned and also to develop species-specific protocols for ART in endangered species (Silva et al. 2004; Roldan et al. 2006). ART developed for the domestic cat facilitated artificial insemination or
RAO ET AL.
in vitro fertilization (IVF) in the puma (Barone et al. 1994), clouded leopard (Howard et al. 1996, 1997), snow leopard (Roth et al. 1997), ocelot (Swanson et al. 1996), cheetah (Howard et al. 1992, 1997) and tiger (Donoghue et al. 1990, 1993a). A similar approach has been used to develop in vitro maturation (IVM) and IVF for rescuing oocytes from various rare felids and nondomestic cat species (Johnston et al. 1991; Pope et al. 1993; Jewgenow et al. 1997). However, the overall efficiency of in vitro embryo production in felid species related to IVM remains inconsistent (Sananmuang et al. 2010), and this inconsistency in the IVM rate could be attributed to several factors such as reproductive status (Donoghue et al. 1993b), culture conditions (Wolfe and Wildt 1996; Pope et al. 1997; Gomez et al. 2003; Hribal et al. 2012; Luu et al. 2013) and the quality of the cumulus-oocyte complexes (Wood and Wildt 1997; Sananmuang et al. 2010). To date, very few attempts have been made to collect oocytes from the ovaries of females of rare felid species which die or undergo medical ovariohysterectomy and to evaluate the potential of such rescued immature oocytes to mature and develop following fertilization in vitro (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007). The present study aims to explore the possibility of oocyte recovery and IVM of oocytes collected from the postmortem ovaries of Indian tiger, Asiatic lion and Indian leopard.
Materials and Methods All the media, hormones and other chemicals used were purchased from Sigma Chemical Co., St. Louis, MO, and plastic ware was from Nunc, Roskilde, Denmark. Collection of ovaries. A total of 16 ovaries were collected from two tigers, three lions and three leopards 6–12 h after death at the Nehru Zoological Park, Hyderabad during the period from November 2009 to September 2013. Ovaries were excised aseptically from the animals at postmortem, washed extensively in sterile Dulbecco’s phosphate-buffered saline (D-PBS; Gibco, Invitrogen Corporation, Carlsbad, CA) containing 100 IU/ml penicillin and 0.1 mg/ml streptomycin and transported to the laboratory within 2 h in prewarmed (37°C) D-PBS. Collection of oocytes. The ovaries (Fig. 1a) were trimmed off adjacent ovarian ligaments and examined for the presence of visible follicles and corpora lutea. Ovarian dimensions (length, width and thickness) were measured with a Vernier calliper, and the weight was also recorded. Each ovary was rinsed once in 70% alcohol for 30 s and three times in prewarmed (37°C) D-PBS and then transferred to a 60-mm culture dish containing 5 ml of filter-sterilized oocyte collection medium (HEPES-buffered tissue culture medium 199
supplemented with 0.3% bovine serum albumin (BSA), 100 IU/ml penicillin, 0.1 mg/ml streptomycin). Each ovary was then processed individually. Incisions were made along the ovarian surface using a sterile surgical blade to release the cumulus-oocyte complexes (COCs) into the oocyte collection medium. Culture dishes were then kept undisturbed for 5 min, to allow the oocytes to settle and examined for COCs under a stereo-zoom microscope (Nikon, Tokyo, Japan). Isolated COCs were then washed five times in fresh droplets of collection medium. The oocytes (Fig. 1b) with darkly pigmented ooplasm and completely surrounded by at least two layers of cumulus cells were selected for IVM. IVM of cumulus-oocyte complexes. The selected COCs were washed five times in droplets of IVM medium (tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 μg/ml FSH, 6 IU/ml LH, 1 μg/ml 17β-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin) and finally transferred into a 4-well plate containing 600 μl of pre-equilibrated IVM medium. The medium was overlaid with equilibrated mineral oil and cultured in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. Measurement of oocyte diameter. At the end of IVM, COCs were examined for cumulus cell expansion (CCE; Fig. 1c). Subsequently, the oocytes were denuded of the cumulus cells by treating with hyaluronidase (100 IU/ml) for 15 min and repeatedly passing the oocytes through a fire-polished narrow bore glass pipette. The average diameter of each oocyte with zona and without zona (ooplasm) was measured at three different sites of the oocyte after in vitro maturation. Zona thickness was measured as the average thickness of the zona at five different sites for each oocyte. Oocyte diameter and zona thicknesses were then ascertained using the XYClone software (Hamilton Thorne, Inc., Beverly, MA). Evaluation of oocytes following IVM. The denuded oocytes were examined for extrusion of the first polar body (PB; Fig. 1d). Those oocytes that did not extrude the first polar body were evaluated for nuclear status according to the method used by Rao et al. (2011). Briefly, denuded oocytes were fixed and permeabilized for 15 min at room temperature in DPBS supplemented with 3.7% paraformaldehyde and 1% Triton X-100 and then placed in D-PBS containing 0.3% polyvinylpyrrolidone for 15 min at room temperature. The oocytes were subsequently transferred into a small drop of DPBS supplemented with 90% glycerol and 10 μg/ml bisbenzimide (Hoechst 33342) on a glass slide, covered with a cover slip supported with four droplets of Vaseline/paraffin and incubated overnight at 4°C. The oocytes were then examined using a fluorescence microscope (Olympus, Tokyo,
MEIOTIC MATURATION OF WILD FELID OOCYTES
Figure 1 In vitro maturation of oocytes recovered from the ovaries (a) of a dead Indian leopard. On the surface of the ovaries the corpora lutea (black arrow) and follicles (white arrow) are visible. Panels b to h show culture grade oocytes before IVM (b), oocytes with cumulus cell
expansion (c), oocytes with first polar body (arrowhead, d), oocytes stained with Hoechst 33342 depicting germinal vesicle stage (e), germinal vesicle breakdown (f), metaphase I (g) and metaphase II with first polar body (h).
Japan) and classified according to chromatin configuration as germinal vesicle (GV; Fig. 1e), germinal vesicle break down (GVBD; Fig. 1f), metaphase I (MI; Fig. 1g) and metaphase II (MII; Fig. 1h). Nuclear maturation was defined as the extrusion of PB and presence of MII spindle in the oocytes. Oocytes that were arrested at the GV, GVBD stage or progressed to MI were considered as immature. Those oocytes with diffusely stained cytoplasm and in which chromatin was fragmented or unidentifiable or not visible were considered as degenerated.
1.5±0.1, 0.9±0.1 and 1.5±0.2, respectively, for tiger; 2.9± 0.2, 1.6±0.1, 1.1±0.1 and 2.8±0.3, respectively, for lion and 2.4±0.3, 1.4±0.1, 0.8±0.1 and 2.3±0.2, respectively, for leopard. The average number of visible follicles per ovary present on the surface of ovaries was 1.0±0.4, 2.8±1.0 and 6.8±2.3 for tiger, lion and leopard, respectively. The mean diameter of the oocytes with and without zona was similar in tiger (169.4±1.9 μm and 126.3±1.6 μm), lion (168.8±1.7 μm and 124.7±1.7 μm) and leopard (176.3± 1.4 μm and 124.2±1.6 μm) and the zona thickness of tiger (20.1±0.3) lion (20.2±0.4) and leopard (21.5±0.3) oocytes were similar. Overall, 119 (average of 14.9 per ovary) oocytes that were collected from postmortem ovaries of 8 donors belong to three different wild felid species (Table 1). The average oocyte recovery was 11.0±5.0 in tiger, 11.0±3.5 in lion and 21.3± 8.8 in leopard. The proportion of good, fair, poor and culture grade oocytes were similar in tiger, lion and leopard (P>0.05). The good and fair quality oocytes were considered as culture grade oocytes and were used for in vitro maturation studies. The mean oocyte recovery was not significantly lower in the animals above 15 yr (8.0±3.1) than the animals below 15 yr (19.0±5.1). Overall, 88 oocytes from individuals of tiger, lion and leopard were subjected for in vitro maturation and their ability to reach different nuclear maturation stages was presented in Table 2. The proportion of oocytes showing CCE, extrusion of PB and matured to different stages of development such as
Statistical analysis. Data was analysed by using SPSS software version 16.0 (SPSS, Inc., Chicago, IL). Oocyte morphometry was analysed by ANOVA. The data related to oocyte quality and the in vitro maturation of oocytes were analysed by chi-square analysis. Differences in means or percentages were considered significant at P15 yr) were analysed by Student’s t test.
Results A total of 16 big cat ovaries (tiger, 4; lion, 6 and leopard, 6) were collected at postmortem. The average length (cm), width (cm), thickness (cm) and weight (g) of ovaries were 2.2±0.1,
RAO ET AL. Table 1 Recovery of oocytes from the ovaries of tiger, lion and leopard collected postmortem Species
Age (years)
Tiger 1 Tiger 2c Total Lion 1 Lion 2 Lion 3c Total Leopard 1b Leopard 2b Leopard 3 Total Overall total (mean±SE) a
15 25
Total number of oocytes recovered (mean±SE)
16 6 22 (11.0±5.0) 14 15 4 33 (11.0±3.5) 39 11 14 64 (21.3±8.8) 119 (14.9±3.8)
22 15 26 15 14 15
Quality of oocytes (%)a Good
Fair
Poor
Culture grade
9 2 11 (50.0) 8 9 1 18 (54.5) 17 5 7 29 (45.3) 58 (7.2±1.7)
4 1 5 (22.7) 4 3 1 8 (24.2) 11 3 3 17 (26.6) 30 (3.8±1.1)
3 3 6 (27.3) 2 3 2 7 (21.2) 11 3 4 18 (28.1) 31 (3.9±1.0)
13 3 16 (72.7) 12 12 2 26 (78.8) 28 8 10 46 (71.9) 88 (11.0±2.8)
No significant differences were found in the proportion of oocyte quality in the three big cat species (P>0.05)
b
Pyometra
c
Cystic ovaries
those that exhibited presence of GV, GVBD, MI, MII and degeneration were similar (P>0.05) in all three species. The proportion of the culture grade oocytes, designated CCE, GV, GVBD, MI, MII and degenerated were similar in between two age groups (below 15 yr and above 15 yr, respectively). However, the proportion of oocytes extruding PB was significantly higher in those oocytes collected from the animals of below 15 yr of age (Fig. 2). Those oocytes extruding polar bodies were vitrified using cryotop method as described earlier (Rao et al. 2012).
Discussion Due to the limited availability and accessibility of gametes from wild felids, very little information is available regarding collection, preservation and culture of the gametes and this is all the more true with respect to the female gametes when compared to the male gametes. Domestic cat has served as convenient research models for wild felid conservation programmes since ARTs optimized using these gametes were
directly applied to the gametes of wild species (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007; Nestle et al. 2012). For instance, the development of reliable ART for domestic cat facilitated the successful artificial insemination and in vitro fertilization (IVF) in the puma (Barone et al. 1994), clouded leopard (Howard et al. 1996, 1997), snow leopard (Roth et al. 1997), ocelot (Swanson et al. 1996), cheetah (Howard et al. 1992, 1997) and tiger (Donoghue et al. 1990, 1993a). A similar approach has also been used to evaluate the development potential of rescuing oocytes from the ovaries of various rare felid species which die or undergo medical ovariohysterectomy (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007). In the present study, attempts were made to study the meiotic competence of rescued oocytes collected postmortem from the ovaries of the tiger, Asiatic lion and Indian leopard after in vitro maturation. In the present study, we demonstrated that it is possible to recover oocytes from the postmortem ovaries of lion, tiger and leopard and matured in vitro, irrespective to their age and reproductive health status. However, those animals above
Table 2 Nuclear status of the felid oocytes after in vitro maturation Species
Tiger Lion Leopard
Number of oocytes
16 26 46
CCE
11 (68.8) 18 (69.2) 33 (71.3)
Oocytes at different maturation status (%) GV
GVBD
MI
TI/M II
PB
Degenerated
Total matured (TI/MII+PB)
1 (6.3) 2 (7.7) 2 (4.3)
2 (12.5) 3 (11.5) 5 (10.9)
3 (18.8) 5 (19.2) 9 (19.6)
5 (31.3) 8 (30.8) 15 (32.6)
4 (25.0) 6 (23.1) 12 (26.1)
1 (6.3) 2 (7.7) 3 (6.5)
9 (56.3) 14 (53.8) 27 (58.7)
MEIOTIC MATURATION OF WILD FELID OOCYTES Figure 2 Effect of age on quality of the oocytes recovered and their meiotic competence after in vitro maturation. Means with different superscripts differs significantly (P15 yr) than the young animals (
Meiotic maturation of oocytes recovered from the ovaries of Indian big cats at postmortem Brahmasani Sambasiva Rao & Yelisetti Uma Mahesh & Komjeti Suman & Katari Venu Charan & Rhisita Nath & K Ramachander Rao
Received: 25 March 2014 / Accepted: 22 July 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract In order to increase the available sources of genetic material for endangered members of the great cat family, this study was designed to assess the meiotic competence of oocytes recovered from postmortem ovaries of the Indian leopard, tiger and lion. The average number of oocytes that were recovered per ovary was 11.0±5.0, 11.0±3.5 and 21.3± 8.8 for tiger, lion and leopard, respectively. The proportion of culture grade oocytes for tiger, lion and leopard were 72.7, 78.8 and 71.9%, respectively. The culture grade oocytes were matured in tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 μg/ml FSH, 6 IU/ml LH, 1 μg/ml 17β-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. After in vitro maturation, 56.3, 53.8 and 58.7% of the tiger, lion and leopard oocytes, respectively, were matured. The proportion of oocytes that extruded first polar body was significantly higher when the oocytes were collected from the animals of less than 15 yr of age compared to above 15 yr. These findings suggest that the oocytes recovered from ovaries of tiger, lion and leopard immediately postmortem can be successfully matured to MII stage.
Keywords In vitro maturation . Oocyte . Leopard . Tiger . Lion
B. S. Rao (*) : Y. U. Mahesh : K. Suman : K. V. Charan : R. Nath : K. R. Rao Laboratory for Conservation of the Endangered Species (LaCONES), CSIR—Centre for Cellular and Molecular Biology (CSIR-CCMB), Uppal Road, Hyderabad 500 007, India e-mail: [email protected]
Introduction The cat family (Felidae) is represented by 37 species of which all members except the domestic cat are classified as threatened, vulnerable or endangered (Nowell and Jackson 1996). The Asiatic lion (Panthera leo persica), Indian tiger (Panthera tigris tigris) and Indian leopard (Panthera pardus fusca) are considered as Schedule I (endangered) animals by the Indian Wild Life Protection Act (1972) due to their decline in population in alarming numbers. The reasons attributed for their decline are habitat loss, depletion of prey base, competition from other predators and poaching, and range decline is considered a strong indicator of population decline (Dinerstein et al. 2007). The International Union for Conservation of Nature (IUCN) has classified the Asiatic lion and the Indian tiger as endangered and the Indian leopard as near threatened (IUCN 2014). In order to increase the population size of the wild/endangered species, apart from management practices, application of assisted reproductive technologies (ART) would be necessary for the success of captive breeding programmes (Wildt et al. 1986; Pope 2000; Holt et al. 2004; Swanson 2006; Andrabi and Maxwell 2007). However, the application of ART for the propagation of wild/endangered species is limited by several factors (Pukazhenthi et al. 2006; Roldan et al. 2006), such as the limited number of animals and lack of availability of species-specific biological material required for a better understanding of the fundamental biology of gametes (Wildt et al. 1986). It is in this context that recovery of gametes from wild/endangered animals which have died due to accidents, medical reasons, disease outbreaks and old age could serve as a resource, to understand the fundamental physiology of the species concerned and also to develop species-specific protocols for ART in endangered species (Silva et al. 2004; Roldan et al. 2006). ART developed for the domestic cat facilitated artificial insemination or
RAO ET AL.
in vitro fertilization (IVF) in the puma (Barone et al. 1994), clouded leopard (Howard et al. 1996, 1997), snow leopard (Roth et al. 1997), ocelot (Swanson et al. 1996), cheetah (Howard et al. 1992, 1997) and tiger (Donoghue et al. 1990, 1993a). A similar approach has been used to develop in vitro maturation (IVM) and IVF for rescuing oocytes from various rare felids and nondomestic cat species (Johnston et al. 1991; Pope et al. 1993; Jewgenow et al. 1997). However, the overall efficiency of in vitro embryo production in felid species related to IVM remains inconsistent (Sananmuang et al. 2010), and this inconsistency in the IVM rate could be attributed to several factors such as reproductive status (Donoghue et al. 1993b), culture conditions (Wolfe and Wildt 1996; Pope et al. 1997; Gomez et al. 2003; Hribal et al. 2012; Luu et al. 2013) and the quality of the cumulus-oocyte complexes (Wood and Wildt 1997; Sananmuang et al. 2010). To date, very few attempts have been made to collect oocytes from the ovaries of females of rare felid species which die or undergo medical ovariohysterectomy and to evaluate the potential of such rescued immature oocytes to mature and develop following fertilization in vitro (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007). The present study aims to explore the possibility of oocyte recovery and IVM of oocytes collected from the postmortem ovaries of Indian tiger, Asiatic lion and Indian leopard.
Materials and Methods All the media, hormones and other chemicals used were purchased from Sigma Chemical Co., St. Louis, MO, and plastic ware was from Nunc, Roskilde, Denmark. Collection of ovaries. A total of 16 ovaries were collected from two tigers, three lions and three leopards 6–12 h after death at the Nehru Zoological Park, Hyderabad during the period from November 2009 to September 2013. Ovaries were excised aseptically from the animals at postmortem, washed extensively in sterile Dulbecco’s phosphate-buffered saline (D-PBS; Gibco, Invitrogen Corporation, Carlsbad, CA) containing 100 IU/ml penicillin and 0.1 mg/ml streptomycin and transported to the laboratory within 2 h in prewarmed (37°C) D-PBS. Collection of oocytes. The ovaries (Fig. 1a) were trimmed off adjacent ovarian ligaments and examined for the presence of visible follicles and corpora lutea. Ovarian dimensions (length, width and thickness) were measured with a Vernier calliper, and the weight was also recorded. Each ovary was rinsed once in 70% alcohol for 30 s and three times in prewarmed (37°C) D-PBS and then transferred to a 60-mm culture dish containing 5 ml of filter-sterilized oocyte collection medium (HEPES-buffered tissue culture medium 199
supplemented with 0.3% bovine serum albumin (BSA), 100 IU/ml penicillin, 0.1 mg/ml streptomycin). Each ovary was then processed individually. Incisions were made along the ovarian surface using a sterile surgical blade to release the cumulus-oocyte complexes (COCs) into the oocyte collection medium. Culture dishes were then kept undisturbed for 5 min, to allow the oocytes to settle and examined for COCs under a stereo-zoom microscope (Nikon, Tokyo, Japan). Isolated COCs were then washed five times in fresh droplets of collection medium. The oocytes (Fig. 1b) with darkly pigmented ooplasm and completely surrounded by at least two layers of cumulus cells were selected for IVM. IVM of cumulus-oocyte complexes. The selected COCs were washed five times in droplets of IVM medium (tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 μg/ml FSH, 6 IU/ml LH, 1 μg/ml 17β-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin) and finally transferred into a 4-well plate containing 600 μl of pre-equilibrated IVM medium. The medium was overlaid with equilibrated mineral oil and cultured in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. Measurement of oocyte diameter. At the end of IVM, COCs were examined for cumulus cell expansion (CCE; Fig. 1c). Subsequently, the oocytes were denuded of the cumulus cells by treating with hyaluronidase (100 IU/ml) for 15 min and repeatedly passing the oocytes through a fire-polished narrow bore glass pipette. The average diameter of each oocyte with zona and without zona (ooplasm) was measured at three different sites of the oocyte after in vitro maturation. Zona thickness was measured as the average thickness of the zona at five different sites for each oocyte. Oocyte diameter and zona thicknesses were then ascertained using the XYClone software (Hamilton Thorne, Inc., Beverly, MA). Evaluation of oocytes following IVM. The denuded oocytes were examined for extrusion of the first polar body (PB; Fig. 1d). Those oocytes that did not extrude the first polar body were evaluated for nuclear status according to the method used by Rao et al. (2011). Briefly, denuded oocytes were fixed and permeabilized for 15 min at room temperature in DPBS supplemented with 3.7% paraformaldehyde and 1% Triton X-100 and then placed in D-PBS containing 0.3% polyvinylpyrrolidone for 15 min at room temperature. The oocytes were subsequently transferred into a small drop of DPBS supplemented with 90% glycerol and 10 μg/ml bisbenzimide (Hoechst 33342) on a glass slide, covered with a cover slip supported with four droplets of Vaseline/paraffin and incubated overnight at 4°C. The oocytes were then examined using a fluorescence microscope (Olympus, Tokyo,
MEIOTIC MATURATION OF WILD FELID OOCYTES
Figure 1 In vitro maturation of oocytes recovered from the ovaries (a) of a dead Indian leopard. On the surface of the ovaries the corpora lutea (black arrow) and follicles (white arrow) are visible. Panels b to h show culture grade oocytes before IVM (b), oocytes with cumulus cell
expansion (c), oocytes with first polar body (arrowhead, d), oocytes stained with Hoechst 33342 depicting germinal vesicle stage (e), germinal vesicle breakdown (f), metaphase I (g) and metaphase II with first polar body (h).
Japan) and classified according to chromatin configuration as germinal vesicle (GV; Fig. 1e), germinal vesicle break down (GVBD; Fig. 1f), metaphase I (MI; Fig. 1g) and metaphase II (MII; Fig. 1h). Nuclear maturation was defined as the extrusion of PB and presence of MII spindle in the oocytes. Oocytes that were arrested at the GV, GVBD stage or progressed to MI were considered as immature. Those oocytes with diffusely stained cytoplasm and in which chromatin was fragmented or unidentifiable or not visible were considered as degenerated.
1.5±0.1, 0.9±0.1 and 1.5±0.2, respectively, for tiger; 2.9± 0.2, 1.6±0.1, 1.1±0.1 and 2.8±0.3, respectively, for lion and 2.4±0.3, 1.4±0.1, 0.8±0.1 and 2.3±0.2, respectively, for leopard. The average number of visible follicles per ovary present on the surface of ovaries was 1.0±0.4, 2.8±1.0 and 6.8±2.3 for tiger, lion and leopard, respectively. The mean diameter of the oocytes with and without zona was similar in tiger (169.4±1.9 μm and 126.3±1.6 μm), lion (168.8±1.7 μm and 124.7±1.7 μm) and leopard (176.3± 1.4 μm and 124.2±1.6 μm) and the zona thickness of tiger (20.1±0.3) lion (20.2±0.4) and leopard (21.5±0.3) oocytes were similar. Overall, 119 (average of 14.9 per ovary) oocytes that were collected from postmortem ovaries of 8 donors belong to three different wild felid species (Table 1). The average oocyte recovery was 11.0±5.0 in tiger, 11.0±3.5 in lion and 21.3± 8.8 in leopard. The proportion of good, fair, poor and culture grade oocytes were similar in tiger, lion and leopard (P>0.05). The good and fair quality oocytes were considered as culture grade oocytes and were used for in vitro maturation studies. The mean oocyte recovery was not significantly lower in the animals above 15 yr (8.0±3.1) than the animals below 15 yr (19.0±5.1). Overall, 88 oocytes from individuals of tiger, lion and leopard were subjected for in vitro maturation and their ability to reach different nuclear maturation stages was presented in Table 2. The proportion of oocytes showing CCE, extrusion of PB and matured to different stages of development such as
Statistical analysis. Data was analysed by using SPSS software version 16.0 (SPSS, Inc., Chicago, IL). Oocyte morphometry was analysed by ANOVA. The data related to oocyte quality and the in vitro maturation of oocytes were analysed by chi-square analysis. Differences in means or percentages were considered significant at P15 yr) were analysed by Student’s t test.
Results A total of 16 big cat ovaries (tiger, 4; lion, 6 and leopard, 6) were collected at postmortem. The average length (cm), width (cm), thickness (cm) and weight (g) of ovaries were 2.2±0.1,
RAO ET AL. Table 1 Recovery of oocytes from the ovaries of tiger, lion and leopard collected postmortem Species
Age (years)
Tiger 1 Tiger 2c Total Lion 1 Lion 2 Lion 3c Total Leopard 1b Leopard 2b Leopard 3 Total Overall total (mean±SE) a
15 25
Total number of oocytes recovered (mean±SE)
16 6 22 (11.0±5.0) 14 15 4 33 (11.0±3.5) 39 11 14 64 (21.3±8.8) 119 (14.9±3.8)
22 15 26 15 14 15
Quality of oocytes (%)a Good
Fair
Poor
Culture grade
9 2 11 (50.0) 8 9 1 18 (54.5) 17 5 7 29 (45.3) 58 (7.2±1.7)
4 1 5 (22.7) 4 3 1 8 (24.2) 11 3 3 17 (26.6) 30 (3.8±1.1)
3 3 6 (27.3) 2 3 2 7 (21.2) 11 3 4 18 (28.1) 31 (3.9±1.0)
13 3 16 (72.7) 12 12 2 26 (78.8) 28 8 10 46 (71.9) 88 (11.0±2.8)
No significant differences were found in the proportion of oocyte quality in the three big cat species (P>0.05)
b
Pyometra
c
Cystic ovaries
those that exhibited presence of GV, GVBD, MI, MII and degeneration were similar (P>0.05) in all three species. The proportion of the culture grade oocytes, designated CCE, GV, GVBD, MI, MII and degenerated were similar in between two age groups (below 15 yr and above 15 yr, respectively). However, the proportion of oocytes extruding PB was significantly higher in those oocytes collected from the animals of below 15 yr of age (Fig. 2). Those oocytes extruding polar bodies were vitrified using cryotop method as described earlier (Rao et al. 2012).
Discussion Due to the limited availability and accessibility of gametes from wild felids, very little information is available regarding collection, preservation and culture of the gametes and this is all the more true with respect to the female gametes when compared to the male gametes. Domestic cat has served as convenient research models for wild felid conservation programmes since ARTs optimized using these gametes were
directly applied to the gametes of wild species (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007; Nestle et al. 2012). For instance, the development of reliable ART for domestic cat facilitated the successful artificial insemination and in vitro fertilization (IVF) in the puma (Barone et al. 1994), clouded leopard (Howard et al. 1996, 1997), snow leopard (Roth et al. 1997), ocelot (Swanson et al. 1996), cheetah (Howard et al. 1992, 1997) and tiger (Donoghue et al. 1990, 1993a). A similar approach has also been used to evaluate the development potential of rescuing oocytes from the ovaries of various rare felid species which die or undergo medical ovariohysterectomy (Johnston et al. 1991; Jewgenow et al. 1997; Merlo et al. 2006; Adamiak and Bartels 2007). In the present study, attempts were made to study the meiotic competence of rescued oocytes collected postmortem from the ovaries of the tiger, Asiatic lion and Indian leopard after in vitro maturation. In the present study, we demonstrated that it is possible to recover oocytes from the postmortem ovaries of lion, tiger and leopard and matured in vitro, irrespective to their age and reproductive health status. However, those animals above
Table 2 Nuclear status of the felid oocytes after in vitro maturation Species
Tiger Lion Leopard
Number of oocytes
16 26 46
CCE
11 (68.8) 18 (69.2) 33 (71.3)
Oocytes at different maturation status (%) GV
GVBD
MI
TI/M II
PB
Degenerated
Total matured (TI/MII+PB)
1 (6.3) 2 (7.7) 2 (4.3)
2 (12.5) 3 (11.5) 5 (10.9)
3 (18.8) 5 (19.2) 9 (19.6)
5 (31.3) 8 (30.8) 15 (32.6)
4 (25.0) 6 (23.1) 12 (26.1)
1 (6.3) 2 (7.7) 3 (6.5)
9 (56.3) 14 (53.8) 27 (58.7)
MEIOTIC MATURATION OF WILD FELID OOCYTES Figure 2 Effect of age on quality of the oocytes recovered and their meiotic competence after in vitro maturation. Means with different superscripts differs significantly (P15 yr) than the young animals (
