1. Zoo Biology 32: 172–176 (2013)
RESEARCH ARTICLE
Parthenogenesis in a Brazilian Rainbow Boa
(Epicrates cenchria cenchria)
Matthew E. Kinney,1,2∗ Raymund F. Wack,2,3 Robert A. Grahn,4 and Leslie Lyons4
1
William R. Pritchard Veterinary Medical Teaching Hospital, University of California-Davis, Davis, California
CA
Sacramento Zoo, Sacramento, CA
California
3
Wildlife Health Center, University of California Davis, Davis, CA
California
4
Department of Population Health and Reproduction, University of California-Davis, Davis, California
CA
2
A 22-year-old captive Brazilian rainbow boa (Epicrates cenchria cenchria) gave birth to four offspring after being
housed with a vasectomized male. Sexual reproduction as a result of failed prior vasectomy, recanalization of the vas
deferens, or prolonged sperm storage was ruled out using the clinical history, histopathology, and gross necropsy. Short
tandem repeat (STR) DNA markers were genotyped in the male, female, and four offspring. None of the offspring
possessed a diagnostic STR allele present in the potential sire. In addition, all offspring were homozygous at each STR
locus evaluated, supporting parthenogenetic reproduction. This is the first report of parthenogenesis in a Brazilian
rainbow boa and has implications for the conservation of reptiles maintained in captive breeding programs. Zoo Biol.
Biol.
32:172–176, 2013. C 2012© 2012Periodicals, Inc. Inc.
00:1–5, 2012.
Wiley Wiley Periodicals,
Keywords: automixis; microsatellite; parentage; sperm storage; vasectomy
INTRODUCTION
MATERIALS AND METHODS
A 22-year-old captive Brazilian rainbow boa
(Epicrates cenchria cenchria) gave birth to four snakes after being housed with a male rainbow boa which had
been vasectomized 59 months prior. A single snake was
still born. The remaining three snakes have continued to
thrive during the 36 months since their birth. At the time
of birth, four explanations were considered to elucidate
the births; incomplete vasectomy, recanalization of the vas
deferens, prolonged sperm storage, and parthenogenetic
reproduction. To determine whether offspring were produced through sexual reproduction or parthenogenesis,
DNA was isolated from the male, female, and all four offspring. Analysis of short tandem repeat (STR) markers
demonstrated the offspring were the result of parthenogenetic cloning. Here, we explain how progeny production from incomplete vasectomy, recanalization of the
vas deferens, and prolonged sperm storage were ruled
out and provide evidence to support parthenogenetic
reproduction.
Review of Specimen History
© 2012 Wiley Periodicals, Inc.
The adult male and female Brazilian rainbow boas
in this report were purchased at 3 months of age from a
private facility and housed together in a zoological collection. These snakes were captive bred and since 3 months
of age they have been housed as a pair with no other animals. The female snake gave birth to viable offspring four
times prior to the male’s vasectomy. Multiple observations of copulation were noted before and after the male’s
vasectomy while the pair remained in the same enclosure.
*Correspondence to: Matthew E. Kinney, Sacramento Zoo, 3930 West
∗ Correspondence to: Matthew E. Kinney, Sacramento Zoo, 3930 West
Land Park Drive, Sacramento, CA 95822. E-mail: mkinney@ucdavis.
edu
Land Park Drive, Sacramento, CA 95822. E-mail: mkinney@ucdavis.
edu
Received 11 June 2012; Revised 11 September 2012; Accepted 21 September
2012
Received 11 June 2012; Revised 11 September 2012; Accepted 21 September 2012
DOI 10.1002/zoo.21050
Published online 19
DOI 10.1002/zoo.21050 October 2012 in Wiley Online Library
(wileyonlinelibrary.com).
Published online in Wiley Online Library (wileyonlinelibrary.com).

2. Parthenogenesis in a Brazilian Rainbow Boa 173
No DNA was collected from offspring produced prior to
vasectomy. Twenty-seven months following vasectomy of
the male, the female gave birth to two live snakes. DNA
was not isolated from these two snakes. Offspring (three
live and one stillborn) were born 59 months following vasectomy of the male. The three viable offspring were determined to be female using cloacal probing at 30 months of
age. Briefly, a probe was inserted caudally into the cloacal
opening after sterile lubrication. In all snakes, the depth of
the probe was equal to or less than three subcaudal scutes,
which is consistent with probe depth previously described
for female rainbow boas (E. cenchria) (three scutes) [Ross
and Marzec, 1990].
The male snake was found dead in the enclosure
16 months after the birth of the four offspring. Gross
necropsy and histopathology revealed the cause of death
to be chronic cardiac disease. Histological examination of
the vas deferens demonstrated fibrosis, complete occlusion, and cystic changes bilaterally. No evidence of vas
deferens recanalization was found after examination of
multiple tissue sections. Marked testicular atrophy and
minimal spermatogenesis was present.
DNA Isolation
DNA was isolated from a whole blood sample obtained from the tail vein of the adult male and female
snakes following the nucleated erythrocyte protocol, using DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia,
CA). A 3-mm segment of tail was collected from the stillborn snake and the first shed skin was collected from the
three viable offspring. A 1-cm segment of shed skin from
each viable offspring was minced and used for DNA isolation. All tissue (one tail tip and three shed skin) samples
from the four offspring were digested overnight following
the tissue extraction protocol of the DNeasy Blood and
Tissue kit. To remove any undigested scales, the digested
tissue was centrifuged prior to application of the solution to the wash column. To compensate for low yield,
DNA isolated from shed skin was concentrated prior to
polymerase chain reaction (PCR) amplification using Genomic DNA Clean and Concentrator Kit (ZYMO Research Corp., Irvine, CA).
STR Amplification
Thirty-nine STR markers previously characterized
in three genera of boids were used for analysis [Matson
et al., 2001; Ramanana et al., 2009; Tzika et al., 2008].
Forward primers were modified with a 5 addition of
a sequence specific to a dye-labeled primer. Using approximately 10 ng of template DNA for each sample, the
following 20 μl PCR reaction conditions were followed:
1 pmol of each dye-labeled primer and STR-specific reverse primer, 0.1 pmol each STR-specific forward primer,
2.0 mM dNTP, 2.0 mM MgCl2 , 0.5% BSA, 1X PCR buffer
II, and 0.375 U AmpliTaq polymerase (Applied Biosys-
tems, Foster City, CA). Each STR was amplified in a
separate reaction using the following temperature profile:
3-min incubation at 94◦ C followed by 35 cycles of denaturation at 94◦ C for 30 sec, annealing at 55◦ C for 20 sec,
and extension at 72◦ C for 45 sec. The amplification was
followed by a final extension step of 30 min at 72◦ C to
ensure that all products were full length. All PCR amplification was performed on a GeneAmp PCR Systems
9700 (Applied Biosystems). Products were individually
size separated on an ABI 3730 DNA analyzer and exact
fragment sizes were determined using STRand analysis
software [Toonen and Hughes, 2001].
RESULTS
Thirty-nine STRs were tested for analysis. Eighteen
STRs failed to produce products or generated nonspecific
products. Twelve were homozygous in all samples and
therefore nondiagnostic. The alleles for STR marker μsat
3 were uninformative for this male-female pairing and the
male shared an allele with the female for μsat 24 that
did not segregate in the offspring. The remaining nine
STRs had diagnostic differences between the two adults
that would have been present in the offspring with proper
segregation (Table 1). All offspring were homozygous for
all STR loci, always an allele present in the female snake.
In no instance did any offspring possess a diagnostic allele
from the sire. Offspring 1 possessed four markers that
displayed only the maternal allele that was not present
in the male, offspring 2 had seven exclusionary markers,
offspring 3 and the nonviable offspring each had three
markers with only a maternal allele.
DISCUSSION
Surgical vasectomy was first reported in snakes (two
garter snakes (Thamnophis sirtalis radix) in 1979 [Zwart
et al., 1979]. In all species, following identification of the
vas deferens, the procedure is relatively simple and involves bilateral removal of a portion of the vas deferens
with ligation or plugging of the remaining vas deferens. If
anatomy is not correctly identified, inadvertent removal
of non-vas deferens tissue is possible, which may result
in continued male fertility. In this case, histopathologic
examination of the tissue removed at the time of the vasectomy verified removal of two 3–4 mm segments of
tissue composed of fibrous tissue with multiple profiles
of tubules lined by pseudostratified columnar epithelium
surrounded by smooth muscles, consistent with vas deferens and epididymis.
Vas deferens recanalization has not been reported in
reptilian species. However, in humans postvasectomy recanalization has been well studied. In general, recanalization is extremely rare and may represent granuloma formation with multiple small proliferating epithelial lined
channels containing spermatozoa [Haldar et al., 2000].
Zoo Biology

3. 174 Kinney et al.
TABLE 1. STR genotypes of potentially parthenogenic Brazilian rainbow boas
STR
μsat 1
μsat 3
μsat 11
μsat 24
55HDZ314
5HDZ277
55HDZ554
55HDZ602
55HDZ617
Male
314
213
274
272A
184A
204
180
214A
218A
318A
229
274
292
184A
208A
182A
214A
220
Female
314
213
274
292
183B
204
176B
204B
220
Offspring 1
364B
229
278B
292
183B
210B
180
212B
222B
314
213
274
292
183B
210B
176B
204B
220
314
213
274
292
183B
210B
176B
204B
220
Offspring 2
364B
213
278B
292
183B
210B
176B
204B
222B
Offspring 3
Offspring 4
(dead)
364B
213
278B
292
183B
210B
176B
204B
222B
314
229
274
292
183B
204
180
212B
222B
314
229
274
292
183B
204
180
212B
222B
364B
213
274
292
183B
204
180
212B
220
364B
213
274
292
183B
204
180
212B
220
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
Loci with no diagnostic value
5HDZ229
5HDZ317
5HDZ360
5HDZ667
55HDZ11
55HDZ220
55HDZ302
55HDZ314
55HDZ339
55HDZ557
55HDZ56
55HDZ600
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
288
164
208
154
222
226
180
184
182
222
368
190
Twenty-one STRs provided robust amplification products for analysis. Bolded types were diagnostically informative. A = female
exclusionary alleles (alleles present only in the male), B = male exclusionary alleles (alleles present only in the female). STR = short
tandem repeats.
In one study of 870 vasectomized men, 50 (5.7%) had
at least one post vasectomy semen analysis that showed
motile sperm, which the authors considered evidence of
recanalization [Labrecque et al., 2003]. It is important
to note that while early (<6 month) or late (>6 month)
recanalization is a considerable risk, the pregnancy rate
after vasectomy is about 1 in 2,000 (0.05%) likely due to
reduced number of sperm in the ejaculate [Philp et al.,
1984]. Upon the death of the potential sire in the case
described in this report, recanalization of the vas deferens with subsequent male fertility was ruled out. Gross
necropsy and histopathological evaluation concluded that
the vas deferens was disrupted with adhesions (to the regional fat pads), fibrosis, and cystic changes bilaterally.
The vas deferens were both occluded and testicular atrophy with minimal spermatogenesis was noted in both
testicles. Therefore, recanalization of the vas deferens following vasectomy was eliminated as a possible mechanism
of reproduction in the case reported here.
Prolonged sperm storage has been documented in a
number of snake species [Birkhead and Møller, 1993; Fox,
1976; Stewart, 1972]. Seven years and six months is the
longest reported time period of suspected sperm storage in
a snake [Magnusson, 1979]. However, in most reports of
suspected prolonged sperm storage, parthenogenetic reproduction was not excluded as a mechanism for offspring
production. A number of hypotheses have been proposed
regarding the function of prolonged sperm storage includ-
Zoo Biology
ing; separation of reproductive events (copulation and fertilization) to optimize the timing in both sexes, insurance
against not finding a partner (especially in species with a
low likelihood of encountering members of the opposite
sex), and enhanced opportunities for sperm competition,
as sperm storage could extend the time period over which
sperm from different males overlap in the reproductive
tract [Birkhead and Møller, 1993]. In the present case, the
male and female were housed in the same enclosure prior
to vasectomy. Vasectomy was performed approximately
five years prior to birth of the four offspring making sperm
storage unlikely. To definitively exclude prolonged sperm
storage, genetic analysis was required to demonstrate the
lack of male genetic contribution to the offspring.
STR genotypes supported offspring parthenogenesis. There were two possible parents but no female exclusionary alleles (alleles present only in the potential
sire) present in any of the offspring. The lack of the male
only alleles and the observed allele homozygosity of the
STR markers in the offspring support derivation from a
parthenogenetic event.
Parthenogenesis has been described in a variety of
vertebrates including captive fish, amphibians, reptiles,
birds, and mammals [Bartelmez and Riddle, 1924; Booth
et al., 2011a, 2011b; Chapman et al., 2007; Feldheim et al.,
2010; Groot et al., 2003; Kono et al., 2004; Lenk et al.,
2005; Olsen and Marsden 1954; Robinson et al., 2011;
Sarvella, 1973; Schut et al., 2008; Smith et al., 2000;

4. Parthenogenesis in a Brazilian Rainbow Boa 175
Spurway, 1953; Watts et al., 2006]. A necessary requirement for viable parthenogenetic reproduction is the maintenance of ploidy levels of offspring. Three mechanisms
have been described to maintain ploidy levels; apomixis,
premeiotic doubling of chromosomes, and automixis
[Lampert, 2008]. In the absence of mutations, apomixis
and premeiotic doubling of chromosomes produces offspring genetically identical to the mother. In the present
case, the heterozygous state of the female is not observed
at any of the STR in the offspring, thus excluding apomixis
and premeiotic doubling. Automixis can produce genetically variable offspring because of segregation and recombination of nonidentical homologous chromosomes.
The degree of variability depends on the exact mechanism
[Lampert, 2008]. Automixis is the only reported mechanism of parthenogenesis in reptilian species [Lampert,
2008]. Apomixis (oocyte production by mitosis) and premeiotic doubling (genome doubling prior to meiosis) in
a Burmese python could not be excluded but was not
proven [Groot et al., 2003]. In the ZW heterogametic sex
determination system, automixis has the potential to result in male or female progeny. If automictic terminal
fusion (fusion of the second polar body with the egg nucleus during meiosis) occurs, male (ZZ) or female (WW)
progeny can result. Until recently, it was thought that female WW vertebrates were nonviable, however a recent
report in a Boa Constrictor (Boa constrictor imperators)
suggests WW females produced from terminal fusion automixis can be viable [Booth et al., 2011a]. In contrast to
terminal fusion, central fusion (fusion of the first polar
body with the oocyte during meiosis) results in female
(ZW) only progeny and maintains maternal heterozygosity [Lampert, 2008]. In the present study, the sex of all
three viable offspring was determined to be female using
cloacal probing. Offspring produced in this case report
are either ZW or WW. Because maternal homozygosity
is maintained in the offspring, automictic terminal fusion
with production of WW progeny is considered most likely.
Parthenogenesis, a rare event with unknown evolutionary significance, has fascinated scientist for a number of years. To the authors’ knowledge, parthenogenetic
reproduction in vertebrates has only been documented
in captive populations with females isolated from males
for an extended period of time. Recently, parthenogenesis has been identified as a possible problem for genetic
management of Komodo dragons (Varanus komodoensis)
[Watts et al., 2006]. Parthenogenisis has the potential to
bias the sex ratio (depending on both the mechanism used
to maintain ploidy levels and gametic sex determination
system of the species) and instantaneously results in homozygosity of the genome, which results in an increased
risk for lower overall fitness [Watts et al., 2006]. Reproductive plasticity has been confirmed in Komodo dragons
using genetic fingerprinting. In a single case, a female was
documented to reproduce asexually through parthenogenesis and later sexually after the introduction of a male.
This observation led to the concern that housing female
Komodo dragons in exhibits without males could induce
parthenogenesis with potential negative impacts on the
population’s genetic pool. The present case is the first to
document parthenogenesis in the presence of a conspecific vasectomized male. This finding is significant as it
can no longer be assumed that the presence of a male prevents parthenogenesis in all species. Unfortunately, the
offspring that were produced prior to vasectomy of the
male are unavailable for genetic analysis and therefore we
can not determine if this female reproduced parthenogenetic offspring while being housed with a sexually capable male. We are also unable to document reproductive plasticity, as the mechanism of reproduction (sexual
vs. parthenogenic) for the offspring produced prior to the
vasectomy of the male was not determined. The documentation of parthenogenesis in an individual housed with a
vasectomized conspecific is important, as it has the potential to influence housing decisions in species that have
breeding recommendations to promote genetic diversity
within a population. Genetic fingerprinting of offspring
produced within populations of endangered species may
be needed to maintain maximum genetic diversity as it
may not be accurate to assume offspring produced by
pairs are the result of sexual reproduction.
CONCLUSIONS
1. Parthenogenesis, most likely as a result of automictic
terminal fusion, was demonstrated for the first time
in the Brazilian rainbow boa (E. cenchria cenchria)
through analysis of STR genotypes.
2. Incomplete vasectomy of the male cagemate, recanalization of the vas deferens, and sperm storage were
excluded using histopathology and genetic analysis.
3. This report of parthenogenesis in an individual housed
with a conspecific male, calls for more complete investigation into the triggers of parthenogenesis and the
potential genetic consequences of parthenogenesis for
captive populations.
REFERENCES
Bartelmez GW, Riddle O. 1924. On parthenogenic cleavage and on
the role of water adsorption on the ovum in the formation of the
subgerminal cavity in the pigeon’s egg. Am J Anat 33:57–66.
Birkhead TR, Møller AP. 1993. Sexual selection and the temporal separation of reproductive events: sperm storage data from reptiles, birds,
and mammals. Biol J Linn Soc 50:295–311.
Booth W, Johnson DH, Moore S, Schal C, Vargo EL. 2011a. Evidence
for viable, non-clonal but fatherless Boa constrictors. Biol Lett 7:253–
256.
Booth W, Million L, Reynolds GR, Burghardt GM, Vargo EL, Schal C,
Tzika AC, Schuett GW. 2011b. Consecutive virgin births in the new
world boid snake, the Colombian Rainbow Boa, Epicrates maurus. J
Heredity 102:759–763.
Chapman DD, Shivji MS, Louis E, Sommer J, Fletcher H, Prodohl P.
2007. Virgin birth in a hammerhead shark. Biol Lett 3:425–427.
Feldheim KA, Chapman DD, Sweet D, Fitzpatrick S, Prodohl PA,
Shivji MS, Snowden B. 2010. Shark virgin birth produces multiple,
viable offspring. J Heredity 101:374–377.
Zoo Biology

5. 176 Kinney et al.
Fox H. 1976. The urinogenital system of reptiles. In: Gans C, Parson TS,
editors. Biology of reptiles, vol 6. London: Academic Press. p 1–157.
Groot TVM, Bruins E, Breeuwer JAJ. 2003. Molecular genetic evidence
for parthenogenesis in the Burmese python, Python molurus bivittatus.
Hereditary 90:130–135.
Haldar N, Cranston D, Turner E, MacKenzie I, Guillebaud J. 2000.
How reliable is a vasectomy? Long-term follow-up of vasectomised
men. Lancet 365:43–44.
Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Sung Park
F, Seo J-S, Ogawa H. 2004. Birth of parthenogenetic mice that can
develop to adulthood. Nature 428:860–864.
Labrecque M, Hoang DQ, Turcot L. 2003. Association between the
length of the vas deferens excised during vasectomy and the risk of
postvasectomy recanalization. Fertil Steril 79:1003–1007.
Lampert KP. 2008. Facultative parthenogenesis in vertebrates: reproductive error or chance? Sex Dev 2:290–301.
Lenk P, Eidenmueller B, Stuadter H, Wicker R, Wink M. 2005. A
parthenogenetic Varanus. Amph Rep 26:507–514.
Magnusson WE. 1979. Production of an embryo by an Acrochoruds
javanicus isolated for years. Copeia 4:744–745.
Matson CW, Williamson JE, Huebinger RM, Louis Jr EE. 2001. Characterization of polymorphic microsatellite loci from the two endemic
genera of Madagascan Boids, Acrantophis and Sanzinia. Mol Ecol
Notes 1:41–43.
Olsen MW, Marsden SJ. 1954. Natural parthenogenesis in turkey eggs.
Science 120:545–546.
Philp T, Guillebaud J, Budd D. 1984. Late failure of vasectomy after
two documented analyses showing azoospermic semen. Brit Med J
289:77–79.
Ramanana MA, Bailey CA, Shore GD, Ramilijaona O, Brenneman RA,
Louis Jr EE. 2009. Characterization of 20 microsatellite marker loci in
Zoo Biology
the Malagasy Tree Boa (Sanzinia madagascariensis madagascariensis).
Conserv Gen 10:1953–1956.
Robinson DP, Baverstock W, Al-Jaru A, Hyland K, Khazanehdari KA.
2011. Annually recurring parthenogenesis in zebra shark Stegostoma
fasciatum. J Fish Bio 79:1376–1382.
Ross RA, Marzec G. 1990. Reproductive husbandry. In: Ross RA,
Marzec G, editors. The reproductive husbandry of pythons and
boas. Stanford, CA: The Institute for Herpetological Research. p 39–
74.
Sarvella P. 1973. Adult parthenogenetic chickens. Nature 243:171.
Schut E, Hemmings N, Birkhead TR. 2008. Parthenogenesis in a
passerine bird, the Zebra Finch Taeniopygia guttata. Ibis 150:197–
199.
Smith HM, Thiss ET, Chiszar D. 2000. Further observation on a
merolepid (partially scaleless) water snake (Nerodia sipedon). Bull
Maryland Herp Soc 36:9–14.
Spurway H. 1953. Spontaneous parthenogenesis in fish. Nature 171:437.
Stewart GR. 1972. An unusual record of sperm storage in a female garter
snake (genus Thamnophis). Herpetologica 28:346–347.
Toonen RJ, Hughes S. 2001. Increased throughput for fragment analysis
on an ABI Prism R 377 Automated Sequencer using a membrane comb
and STRand software. BioTechniques 31:1320–1324.
Tzika AC, Koenig S, Miller R, Garcia G, Remy C, Milinkovitch MC.
2008. Population structure of an endemic vulnerable species, the Jamaican boa (Epicrates subflavus). Mol Ecol 17:533–44.
Watts PC, Buley KR, Sanderson S, Boardman CC, Gibson R. 2006.
Parthenogenesis in Komodo dragons—should males and females be
kept together to avoid triggering virgin birth in these endangered
species? Nature 444:1021–1022.
Zwart P, Dorrestein GM, Stades FC, Broer BH. 1979. Vasectomy in the
garter snake (Thamnophis sirtalis radix). J Zoo An Med 10:17–21.