Vitrification of oocytes from endangered Mexican wolves (Canis lupus baileyi)
S. Boutellea, K. Lenahanb, K. L. Baumana, R. Krisherc, C.S.Asaa*, S. Silberb
a Research Department, Saint Louis Zoo, St. Louis, MO63110United States
bSt. Luke’s Hospital, Chesterfield, MO63017
c University of Illinois, Urbana, IL61801USA
Abstract
1. Introduction
The Mexican gray wolf (Canis lupus baileyi), listed in 1976 as an endangered subspecies of the gray wolf by the U.S. Fish and Wildlife Service (USFWS), provides an excellent example of the successful use of captive breeding in species recovery. Considered extinct in the wild, the current captive population of Mexican wolves numbers about 340 individuals maintained in 47 zoos and related facilities in the U.S. and Mexico (Siminski, 2008). However, because these wolves all trace to only 6 or 7 original founders (Hedrick, 1997), that is, genetic ancestors of the entire captive population, careful genetic management is required. Selection of breeding pairs seeks to maintain or increase gene diversity by considering mean kinship (how many relatives exist in the current population) and avoiding inbreeding (Siminski 2008).
To preserve genes from this limited population, a frozen semen bank was created in 1991 at the Saint Louis Zoo under the auspices of the USFWS Mexican wolf recovery program. At present, this semen bank holds samples from 65 individual Mexican wolves. Until very recently, though, the only method for preserving female gametes was in embryos, a much more complicated and laborious undertaking. In addition, embryos do not permit the genetic flexibility possible with egg and sperm cells, because the genetic match must be made at the time the embryo is created for cryopreservation. The ability to preserve ova from genetically valuable Mexican wolf females for future use in in vitro fertilization would provide an extremely valuable tool to population managers.
Vitrification has been successful with ova from a variety of domestic and laboratory species as well as humans (cow: Hamano et al. 1992; horse and cow: Hurtt et al. 2000; pig; Rojas et al. 2004; cat: Merlo et al 2008; human: Katayama et al. 2003), but has not yet been applied to endangered species recovery programs. Although successful in-vitro maturation and fertilization protocols have not been established for canids, vitrification can rescue genes from female Mexican wolves, especially aging females that have produced few or no offspring, for future use. Given the tremendous advances that have occurred in assisted reproduction and in-vitro culture systems in recent years, and in particular the interest in in-vitro techniques for application to domestic dogs, the probability seems high that methods will be available when the genes represented by these samples might be needed in the future.
The AZA (Association of Zoos and Aquariums) Mexican Wolf SSP (Species Survival Program) and the USFWS Mexican Wolf Recovery Program identified individual female wolves as oocyte donors that because of advancing age or illness were unlikely to reproduce naturally.
2. Materials and methods
2.1. Animals:
Ovaries were obtained from 5 Mexican gray wolves and 1 generic gray wolf (F419) from January through March (the natural breeding season extends from mid-January through mid-March). Two females (Table 1) were stimulated with Ovuplant® (Peptech Animal Health, New South Wales, Australia), which contains 2.1 mg of the GnRH agonist deslorelin, for timed ovulation and oocyte aspiration. Although marketed for ovulation induction in domestic mares, it has been used successfully to induce estrus and ovulation in domestic dogs (Kutzler 2007) and in wolves (Asa et al. 2006). One female (Table 1) had been treated 5 months previously with Suprelorin® (Peptech Animal Health), which also contains deslorelin, but in a slow-release implant matrix used for contraception. Suprelorin first stimulates then down-regulates the reproductive axis. The remaining females were not treated.
The wolves were housed at various zoos and captive breeding facilities in the U.S. (Table 1), which in three cases necessitated shipment of ovaries by air immediately after ovariectomy to our lab in St. Louis. Time from surgery to aspiration following transit ranged from 6 to 8.5 hours. For the other two females, aspiration immediately followed surgery; in one case the team traveled to Minnesota and in the other the female was brought to the Saint Louis Zoo for ovariectomy.
2.2 Shipment of ovaries
Ovaries were left within the bursa for shipment. Immediately after surgical removal, ovaries were wrapped in gauze squares soaked in warm sterile saline, secured in separate ziplock plastic bags, and placed in an 8oz. plastic cup along with a thermometer. The cup was then placed in an Equitainer along with a gel-pack heated to about 38°C. Prior tests with a temperature logger had verified that the temperature was maintained within a few degrees for up to 12 hours. The Equitainer was then shipped same day air cargo to Saint Louis.
2.3 Oocyte recovery
As soon as the shipment arrived in the lab, ovaries were removed from the surrounding bursa with a scalpel, and any structures (e.g., follicles, ovulation sites, corpora lutea) were noted. Aspiration was performed using a vacuum pump (Cook Vmar-5000: Cook Veterinary Products, Bloomington, IN)at 2.8mmHg and 20 gauge needle. The aspirated follicular fluid was collected into a 15ml conical tube containing HEPES buffered medium (Irvine Scientific, Santa Ana, CA).
2.4 Oocyte selection
Follicular fluid with HEPES was transferred into small petri dishes[A1]to[rlk2] prevent clumping and facilitate searching for oocytes. In the first trial with gray wolf 419, to optimize the conditions for successful vitrification, the cumulus cells were removed from the oocytes by exposing them to 60U hyaluronidase for 30 seconds, then cleaning mechanically with pulled glass pipettes. The oocytes were examined for an intact zonapellucida andhomogeneous cytoplasm. Due to the darkness of the cytoplasm, no further morphological assessment was possible, e.g., presence of vacuoles or pits.
The oocytes found were then counted and placed in HEPESmedium on a warm plate. Once all oocytes were located and graded the number to be vitrified was determined. Grading was based upon an intact zona pellucida, homogeneous cytoplasmand compact cumulus cell mass which are consideredto be indicators of oocyte meiotic competence (Rodrigues, 2003). Although the cumulus cell mass can interfere with the vitrification process, these cells appear to be critical to in vitro maturation for dog oocytes (Rodrigues, 2006). Therefore,with oocytes from all Mexican wolves, we leftthe corona cell layer associated with each oocyte intact to improve chances for future maturation and fertilization.
2.5 Vitrification
To prepare for vitrification, each oocyte was exposed to solutions with increasing concentrations of ethylene glycol (Sigma Aldrich, St. Louis, MO) plus DMSO (dimethyl sulfoxide: Sigma Aldrich), starting from 7.5% ethylene glycol and 7.5% DMSO, HEPES medium with 20% Synthetic Serum Substitute (SSS: Irvine Scientific, Santa Ana, CA) to a final exposure to 15% ethylene glycol, 15% DMSO and 0.5M sucrose solution with HEPES and 20% SSS using the method developed by Kuwayama and colleagues (Kuwayama et al. 2000, 2005, Katayama et al. 2003). Oocytes weretransferred from the final vitrification solution to CryoTops (Kitazako Supply Co., Fujinomiya, Japan) and immediately plunged into liquid nitrogen[A3]. CryoTops were then capped and transferred to liquid nitrogen tanks for storage.
2.6 Oocyte thawing and viability testing[rlk4]
Following removal of the protective cap while still submerged in liquid nitrogen, each CryoTop with oocytes to be thawed was transferred directly into a small culture dish containing 5cc thawing solution, consisting of 1M sucrose with HEPES medium and 20% SSS warmed to 37°C. After 1 minute in the thawing solution oocytes were moved through decreasing concentrations of 0.5M sucrose to 0.2M sucrose and finally into a HEPES plus 20% SSS solutionat room temperature.
Immediately post thaw, intact oocytes were stained to assess viability using propidium iodide (PI), or PI with bisbenzimide (Hoechst 33342) (Lee et al., 2006—I need to add a few other references here). Oocytes were placed into D-PBS supplemented with 0.1% polyvinyl alcohol and 100 µg/mL PI and incubated in the dark at room temperature for 15 minutes. Oocytes were examined using a fluorescent microscope. Oocytes stained red (PI positive) due to disruption of the plasma membranes and passage of the PI into the cell indicated a dead oocyte. Viable cells did not stain with any red fluorescence (PI negative; indicative of plasma membrane integrity). Some oocytes were also stained with 10 µg/mL Hoechst in an attempt to visualize chromatin configuration at the same time as viability assessment. However, chromosomes were not clearly seen in many of these oocytes, possibly due to minimal compression of the oocyte at the time of fluorescent examination. Trypan blue staining was also attempted by incubating the oocyte in 0.2% trypan blue in D-PBS for 3-5 minutes, followed by assessment with a light microscope. After staining, oocytes were mounted on a glass slide under a cover slip, fixed in 3:1 acetic acid:ethanol, and stained with orcein to evaluate nuclear maturation status.
3. Results
All ovaries reached our lab between 6 and 8.5 hours of surgery, and a thermometer placed into the bag containing the ovaries verified that the temperature did not drop more than 10 degreesduring shipment. In general, more oocytes were retrieved from the ovaries of females stimulated with the short-acting GnRH agonist Ovuplant, with one notable exception, female 516,whose ovaries yielded the largest number (Table 2). The number of oocytes aspirated per female was not related to the date of collection (i.e., point during the breeding season) nor to female age. The smallest number of oocytes was recovered from the female treated with the long-acting GnRH agonist Suprelorin for contraception the previous October. She appeared to have ovulated following that stimulation, since corpora lutea were identified. However, her reproductive tract had been removed because of pyometra, which also may have affected ovarian dynamics.
Needs statement about oocyte quality before freezing.
Although the trypan blue methods was attempted as a non-fluorescent assay to assess viability, the high lipid content in wolf oocyte cytoplasm, which results in a very dark appearing oocyte, made it difficult to ascertain the level of blue staining and determine viability. Thus, the trypan blue assay was abandoned and the PI/Hoescht assay utilized for all oocytes. A total of 29 oocytes were thawed to assess viability after vitrification and thawing, from three different females (one grey wolf and 2 Mexican wolves). In all, 29 oocytes were thawed. Of these, 8 lysed during the thawing process, leaving 21 intact oocytes for analysis (Table 3). There was no difference in the percentage of intact oocytes post thaw between females (P>0.05). Of the intact oocytes, 9 (42.9%) were graded as PI positive (dead), while 12 (57.1%) were PI negative (alive) (Table 3). There was no difference in the percentage of live oocytes between females, either as a percentage of intact oocytes post thaw, or total vitrified oocytes (P>0.05). Orcein stain revealed that all oocytes remained in the immature, germinal vesicle (GV) stage of meiosis, demonstrating that spontaneous nuclear maturation did not occur during the aspiration, vitrification or thawing process.
4. Discussion
There are many reasons for the variability in oocyte quantity recovered, for example, age has been shown to significantly affect follicle and oocyte number in domestic dogs (Songsasen, 2007; Lopes, 2007). Another influence may be the reproductive stage of the female at the time of oocyte recovery. In this study, all procedures were performed during wolf breeding season between January and early March (needs reference).
Analysis of oocyte viability indicates that this method of vitrification results in viable oocytes post-thaw. Approximately 75% of the vitrified oocytes could be successfully recovered post-thaw, and of these almost 60% were viable. Overall, more than 40% of all vitrified oocytes remained viable after cryopreservation and thawing. This is an encouraging result, as it suggests that wolf oocytes may successfully be cryopreserved with the cryo-top technique with a reasonable success. Thus, as the opportunity for oocyte collection presents itself in the near future, oocytes may be collected and safely cryopreserved, yet used for conservation management breeding programs at a later date. At that time, assisted reproductive technologies for canids may be better established and more efficient, allowing gametes of current and future wolves to be utilized at any time for optimal genetic management of the species.
Assessment of oocyte viability using propidium iodide relies on the ability of healthy, intact plasma membranes in live cells to exclude this dye. This method does not provide any information on the ability of these oocytes to complete nuclear maturation, be fertilized in vitro, or develop into an embryo which could potentially be transferred to a surrogate female where it might initiate and sustain pregnancy. Further in vitro testing of these oocytes must establish these developmental competencies after cryo-top vitrification of wolf oocytes. However, we do know that this oocyte vitrification method has successfully led to offspring in other species, including humans (References needed here—Kathy or Sherm, do you have some good human references?) Once in vitro maturation, fertilization and embryo culture has been successfully developed for the domestic dog, these techniques can be applied to both test the efficacy of the technique examined here, as well as utilize these oocytes in the Mexican wolf SSP. In the meantime, however, this technique will allow us to bank female gametes that would otherwise be lost, thus preserving valuable genetics and potentially contributing to the maintenance of high levels of heterozygosity in future Mexican wolf populations.
In summary, the cryo-top technique represents a viable method by which to safely preserve the female gametes of wolves for later use in captive breeding programs for conservation.
References
Asa CS, Bauman K, Callahan P, Bauman J, Volkmann DH, Jochle W. GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in gray wolves (Canis lupus). Theriogenology 2006;66:1778-1782.
Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fert Steril 2003;80:223-224.
Kutzler MA. Estrus induction and synchronization in canids and felids. Theriogenology 2007;68:354-374.
Kuwayama M, Kato O. All round vitrification method for human oocytes and embryos. J Assist Reprod Genet 2000;17:477.
Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod BioMed 2005;11:300-308 [online].
Lee HS, Yin XJ, Kong,IK. Sensitivity of canine oocytes to low temperature. Theriogenology 2006;66:1468-1470.
Lopes G, Sousa M, Luvoni, GC, Rocha A. Recovery rate, morphological quality and nuclear maturity of canine cumulus-oocyte complexes collected from anestrous or diestrous bitches of different ages. Theriogenology 2007;68:821-825.
Rodrigues B, Rodrigues J. Influence of reproductive status on in vitro oocyte maturation in dogs. Theriogenology 2003;60:59-66.
Rodrigues, B, Rodrigues J. Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome. Theriogenology 2006;66:1667-1672.
Songsasen, N. and Wildt, D. E. Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci. 2007; 98: 2-22.
Table 1. Information on age, origin, treatment and procedure dates for female wolves used in the study.
WolfID / Age
(yrs) / Facility location / Treatment / Ovaries shipped / Shipment
time / Date treated / Date aspirated
419 / 10 / WSC / Ovuplant / No / 11 Jan / 21 Jan
435 / 13 / SWREF / Ovuplant / Yes / 29 Jan / 6 Feb
741 / 7 / EPZ / Suprelorin / Yes / 13 Oct / 12 Mar
188 / 12 / WSC / None / Yes / --- / 9 Jan
204 / 11 / WCSRC / None / No / --- / 19 Feb
516 / 11 / NYWCC / None / Yes / 10 Feb
WSC: WildlifeScienceCenter, Forest Lake, MN
SWREC: Southwest Wildlife Rehabilitation and Education Foundation, Scottsdale, AZ
EPZ: El Paso Zoo: El Paso, TX
WCSRC: Wild Canid Survival and ResearchCenter, Eureka, MO
New YorkWolfConservationCenter, Salem, NY
Table 2.
Wolf ID / Structures on ovaries / Ooctyes apirated / Oocytequality[A5]? / Oocytes
virtified
419 / Many visible follicles / 31[A6] / ? / 25
435 / 5 CL / CH per ovary / 47[A7] / ? / 40
741 / 6 CL on one ovary / 4 / 2 good
2 average / 4
188 / Some visible follicles / 11 / 6 good
1 average
3 poor / 10
204 / Some visible follicles / 12 / 8 good
4 poor / 8
516 / Many visible follicles / 73 / All good / 67
CL: corpora lutea
CH: corpora hemorrhagica
Table 3. Viability of vitrified-thawed wolf oocytes determined by propidium iodide staining†. There are no significant differences between females for any of the parameters measured.
Wolf ID / No. oocytes thawed / No. intact oocytes (%)* / No. viable oocytes (%)** / % viable oocytes of total thawed419 / 15 / 11 (73.3 ± 11.8) / 7 (63.6 ± 15.2) / 46.7 ± 13.3
435 / 10 / 7 (70.0 ± 15.3) / 3(42.9 ± 20.0) / 30.0 ± 15.3
188 / 4 / 3 (75.0 ± 25) / 2(66.7 ± 33.0) / 50.0 ± 28.9
Total / 29 / 21 (72.4 ± 8.5) / 12 (57.1 ± 11.1) / 41.4 ± 9.31
* the number of vitrified oocytes thawed was reduced by oocyte lysis immediately post thaw or during staining (6x), mechanical damage during manipulation (1x), or oocyte loss during manipulation (1x).
**percentage live oocytes of thawed, intact oocytes
† percentages are presented as mean ± SEM
1
[A1]Size?
[rlk2]If they are the small 1008 petri dishes they are 35mm, if 1007 60 mm.
[A3]We do still need to reference the original procedure. If we have the reference to the complete procedure, then we don’t have to provide as much detail.
[rlk4]This procedure needs to be explained as well, with the solutions used and time in each solution. Also, Kathy evaluated each oocyte immediately post thaw—I remember that some lysed, that is in the notes. It could be included in the section above and oocyte cryopreservation and thawing.
[A5]Or should we have 3 columns for good, average, poor?
[A6]I don’t see any data in the notes about the quality of the oocytes for these two females.
[A7]Nori did this animal. Should we assume she only vitrified oocytes she considered of good quality?