Minimize crop damage (rat, various bird species) Control predators perceived to threaten livestock or game species (coyote)
Reduce threat of exotic species to native wildlife (rabbit, fox, etc. in Australia)
Decrease possibility of spread of disease to livestock or humans (skunk)
Minimize damage to ecosystems by locally overabundant species (horse, deer, elephant)
Manage zoo animal population sizes (big cats)
*Avoid public relations repercussions of traditional culling methods
*Lower expenses given PR needs
Since 1950s, when research proliferated on natural and synthetic steroids for human contraception Poorly organized - 1st international conference on wildlife contraception not until 1987
Separation of sexes: Useful only in captive animals; not even very effective there Surgical: Castration, vasectomy/tubal ligation, injecting of sclerosing agents
Irreversible Difficult in field; requires catching, performing surgery
High attendant mortality
Time consuming and expensive
Mechanical: Intra-uterine and vaginal devices, vas deferens plugs
Similar problems as surgical, but potentially reversible But also requires species-specific design and individual tailoring
Injectable: Chemicals, hormones, antigen preparations
May or may not require capture, but at minimum requires locating Reversible, but also has limited period of efficacy
Persistence - especially of steroids - in tissues; passed on to 2° consumers (including humans)
Implantable: Mostly hormones
Similar to injectable, but longer acting Biobullet - silicone matrix rod impregnated with synthetic hormone (especially progestins or combinations), protected on entry with cellulose and calcium carbonate casing
Oral: Chemicals, hormones, antigen preparations
Similar to injectable and implantable, but does not require capture Frequent application of large quantities required
May be consumed by non-target species
Viral: Live attenuated viruses genetically engineered to produce antigensÖ
Chemicals In use since 1950s Often plant products, or agricultural chemicals
Work by inhibiting gametogenesis, especially in males
Least likely to be reversible
Most likely to be toxic
1959 - triethylene melamine (TEM) reduced testis size and spermatogenesis in starling, but extremely toxic
1964 ñ Arasan ñ a fungicide/seed disinfectant ñ produced reversible inhibition of ovulation in pigeon
Hormones
In use since 1960s and 1970s (coyote, pigeon, red-winged blackbird, rat, much of modern work on white-tailed deer) Usually steroidal; progestins or combinations with estrogens (occasionally androgens)
Natural or synthetic; latter generally longer-acting but less biodegradable
MANY formulations; 25-30 different commercially available steroid contraceptives available by 1985
Widely used because of high reversibility, low toxicity
Broad biological action (hypothalamus, pituitary, gonads, other reproductive tissues)
Resultant disturbance of natural behavior
Poor biodegradability; readily available to 2° consumers
Oral and injectable require large doses, especially of natural
1963 ñ synthetic estrogen diethylstilbestrol (DES) used to control fertility in red fox, and many species since
mid 1970s ñ oral progestins, especially melengestrol acetate (MGA; one of few approved for use in livestock)
Antigen preparationsÖ
Variety of antigens used Sex hormones, especially GnRH (in both males and females) Decreases FSH and LH secretion ñ atrophy of gonads - infertility Profound effects on behavior
1980s - pig, rat, rabbit, horse
Spermatozoon (specifically sperm head) proteins
Sperm antibodies occasionally natural cause of human infertility Prevent sperm binding/penetrating ovum
Late 1970s, early 1980s ñ being developed for use in red fox and rabbit in Australia
Zona pellucida (ZP) proteins
Solubilized porcine zona pellucida (PZP) vaccine focus of most work; proven effective in feral horses, burros Highly specific ZP antigens, e.g. mouse ZP3
Prevent sperm binding/penetrating ZP, or prevent implantation if fertilization occurs
Placental components (e.g. riboflavin carrier protein)
Generally considered reversible
Lasts until no longer sufficient antibodies to interfere with biological activity of target antigen, usually a year or so Repeated treatment may result in irreversibility
May also require an alarm signal from stressed/damaged cells to trigger immune response; signal may be heat-shock proteins produced and released by cell surfaces in response to heat or other stressor
Made effective by combining with adjuvant, but some adjuvants trigger localized or even systemic reactions
May be oral if capable of involving pharyngeal or small intestinal immune cells
Proteins digested rapidly in stomach, so require protection to pass through to small intestine; enteric coating to slow gastric degradation Attraction and attachment to mucosal surfaces important; a bacterial lectin coat is useful for this, making it mimic a pathogenic bacterium; liposomes (phospholipid and cholesterol membranes)
Oral delivery requires larger dose of vaccine, and has less predictable result
Proteinaceous in nature ñ breaks down when eaten by 2° consumer, so presumably safe for environment
Behavioral effects minimized (but note continued cycling in unfertilized polyestrous females)
Species- and individual-specific response in antibody development
In antibody titer achieved In time until effective titer reached
In time until titer no longer effective
In whether antibody titer has contraceptive effect (e.g. for some species, PZP is too dissimilar structurally to their own ZP)
May genetically engineer harmless virus (e.g. vaccinia virus, ectromelia virus - mousepox) with ability to produce desired antigen like mouse ZP3
Would be exceptionally effective, economic large-scale strategy Species-specificity and attenuation would be VERY important
Not yet used outside of controlled situation
Still largely ineffective after 4 decades; most methods not feasible for free-ranging animals None effective in reducing populations already too large ñ traditional culling still necessary (reproduction not only factor affecting population size, compensatory survivorship important too)
Effects on non-target species, including humans, still largely unknown
Proliferation of human species Direct killing of wildlife (guns, poisons, automobiles) *Habitat destruction
Loss of much suitable area Fragmentation of remaining suitable area
Areas too small to harbor viable populations of various species, because of genetic considerations
inbreeding depression; resultant infertility, susceptibility to disease, inability to adapt to changing conditions genetic drift with unpredictable results ñ esp. fixation of deleterious alleles (FL panther cryptorchidism)
Populations require assistance to survive in long-term; management of wild lands as parks ñ "zoos without fences"
Translocation is a low tech, relatively low cost strategy, but
Danger to animals during shipping Translocated animals often disperse or are killed before reproducing
Animals may be too valuable to their original population to remove
Zoos also interested because of problems breeding captive animals (mate too distant, behavioral problems, incompatibility, health or age)
Experimentation on exposure of horse semen to "winter conditions" in 1776! Strong (eutherian) mammalian bias
Externally-fertilizing Fish
Hormonally primed to force gamete development and maturation using GnRHs, or extracted pituitaries, or OvaPrim ñ commercial preparation of Salmon GnRH and a dopamine blocker (dopamine is a GnRH blocker) Subsequent hatchery-style combination of gametes and rearing of embryos for later stocking
Same methods being applied to endangered amphibians; sure to increase because they are under global threat
Birds ñ little real work done
Reptiles ñ virtually no work done
Estrus synchronization ñ to manipulate femaleís reproductive state to time readiness for techniques PGF2a in ruminants: destruction of CL and decrease in circulating P4 ñ> initiates sequence of endocrine events resulting in ovulation Exogenous gonadotrophins, e.g. PMSG and HCG in felids (induced ovulators): stimulates LH surge by pituitary ñ> LH induces follicle rupture
Superovulation ñ (ultimately) to obtain largest number of viable embryos for use
Quite a range of potentially viable oocytes produced by species in nature - plains viscacha produces several thousand oocytes each cycle, and eventual litter size is result of subsequent death of oocytes and embryos (remember the most common fate of a follicle is atresia) Exogenous gonadotrophins, e.g. PMSG, FSH
Tip development in favor of ovulation instead of atresia
Semen collection ñ to obtain sperm capable of fertilizing ova
Artificial vagina ñ requires tame animals, risks to handler Electroejaculation ñ variable results, generally requires anesthesia, and so must develop proper protocol to avoid urine contamination, retrograde ejaculation
Biopsy or postmortem ñ mature sperm from cauda epididymis; now also having some success with immature sperm from head of epididymis; maybe even sperm precursors
Special note ñ sperm precursor cells transferred into testes of immunodeficient mice ñ have obtained viable rat sperm; this technique would only require single biopsy from donor to produce large # of sperm
Artificial insemination (AI) Very old technique, still most commonly and widely used Anterior vagina ñ most often used because least invasive, but also least effective
Cervix
Uterine ñ often laparoscopic intrauterine insemination works best
Embryo transfer (ET)
Widely applicable in ungulates (cervids, bovids, camelids, ovids); limited application elsewhere Allows recovery of more of donor females genome
Keeps donor female donating, rather than busy in gestation and lactation of young
Eliminates chance of death due to pregnancy or parturition
Can even use different species
So far used in closely related species, such as mouflon to sheep, gaur to domestic cattle For extremely rare species, where every female is important
Problematic
Embryos might not secrete correct factors, or at right times to prevent luteolysis Physiological mismatch possible: endocrine, immunological problems (rejection by female), formation of inadequate placenta
One solution ñ chimaeric embryos
Outermost layer of placenta (in direct apposition to maternal tissues) derived from trophectoderm, which differentiates from the embryoís inner cell mass at the blastocyst stage, and so does NOT form any part of the fetus So, transplant inner cell mass of endangered species into trophectoderm from recipient species
May not solve all problems, as other major constituents of placenta are formed by inner cell mass cells at later stages
Transfer embryos under laparoscopy rather than general anesthesia and surgery
In vitro fertilization (IVF)
Recent adaptation of human procedures to animals 4 phases
oocyte maturation ñ complex nuclear, cytoplasmic and cytoskeletal processes leading to ovulation sperm capacitation ñ complex changes in cell membrane constituents to prepare sperm to penetrate egg
fertilization ñ combination of sperm and egg
(note problems with capacitation and fertilization being overcome by direct intracytoplasmic (ICSI) and subzonal (SUZI) sperm injection)
embryo culture ñ fostering pre-implantation development
Demanding, expensive, sometimes low pregnancy rates for high-tech methods:
Natural reproduction ñ approximately 80% pregnancy rate (near 100% in captive species) AI ñ 50%
ET ñ 65%
IVF - 20%
Cryopreservation ñ the "Frozen Zoo" Zoos found new purpose in conservation of endangered species Some captive species now extinct in wild But what good is a captive species? ("The California Condor vs. National Audubon Society")
Still, in general successful reintroduction is highly unlikely (whooping cranes in FL, red wolves in NC)Ö
ÖAnd impossible if no habitat remains
Zoos have too little space and other resources to maintain genetically viable populations for more than a handful of species
Number of endangered species increasing at exponential rate
Idea that gametes and/or embryos of endangered species could be cryopreserved in lieu of keeping large population
Goal to maintain frozen zoo until human population passes some "demographic winter"
Then wild lands can be rededicated and endangered species will be "reborn" (some criticize as fantasy)
Accidental discovery in 1949 that glycerol protects sperm during freezing
Cryobiological research on erythrocytes, tissue culture cells, stem and yeast cells in 1950s
Now used in many species
Very long-term storage possible (some mouse embryos already stored >> decade), estimated longevity in THOUSANDS of years
Cryoprotectants were key
Physiochemistry - Heat and water transport between intra- and extracellular environment (mammalian embryos approximately 80% water) Phase change from liquid to solid especially important
Cryoprotectants lower temperature at eutectic point (upper limit of salt solubility) and freezing point
Temperature decrease -> Extracellular water removed as ice -> Osmotic pressure gradient develops between concentrated extracellular solution and dilute intracellular solution -> Efflux of water from embryo -> Influx of cryoprotectant
Permeating cryoprotectants: glycerol, dimethyl sulfoxide (DMSO)
Stabilize membrane phospholipids and protein macromolecules Act as solvent for solutes and electrolytes
Reduce onset of ice formation
*Increase viscosity of intracellular compartments, promoting VITRIFICATION (production of solid, laminar "glass" phase) instead of crystallization
Non-permeating cryoprotectants ñ sucrose, dextrose, albumin
Dehydrate embryonic blastomeres during cooling, thereby decreasing risk of intracellular ice High molarity cryoprotectants best for vitrification
At least one, VS3a, can be used at room temperature
Need to avoid osmotic trauma or cryoprotectant toxicity
Asynchronous development ñ embryo death (recall maternal recognition of pregnancy ñ embryo prevents destruction of CL; CL IMPERATIVE to embryoís early survival) Primates ñ placenta releases luteotrophic gonadotrophin
Ruminants ñ embryo releases interferon before implantation, suppressing luteolysis
Luteal lifespan supplementation with interferon successful in some ungulates
Still much work needed
Complex and still poorly understood interplay between intra-, inter- and extraovarian regulatory mechanisms Many adverse treatment (especially anesthetic) effects, on essentially every stage of development: follicular, oocyte, embryo
All techniques still only with sporadic successes, often not replicated despite effort