Observations on the Spermatogenic Cycle of the Grass Snake, Natrix natrix (Serpentes: Colubridae) in Northern Iran

Asian Herpetological Research 2011, 2(1): 55-59
DOI: 10.3724/SP.J.1245.2011.00055

Observations on the Spermatogenic Cycle of the Grass Snake,
Natrix natrix (Serpentes: Colubridae) in Northern Iran

Afshin FAGHIRI', Abdolhossein SHIRAVI', Vida HOJATI" and Haji Gholi KAMI?

' Department of Biology, Islamic Azad University, Damghan Branch, Damghan, Iran
> Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran

Abstract The Grass Snake, Natrix natrix, is one of the non-venomous oviparous snakes of the family Colubridae,
found in Mazandaran, a northern province of Iran. In order to study the spermatogenic cycle of this species in the
area, sampling was done from April to November, and twenty-six male snakes were collected by hand during daylight
hours. Testes were extracted from the specimens, and were fixed and prepared for histological examination. The results
indicated that the spermatogenesis started in early June, reached its height in August and September, and ended in
November. The mean body length of the specimens was measured 636.21 mm, and the mean volume of testes was
524.54 mm° and 569.06 mm’ for the right and left ones, respectively, and the mean weight of the testes was 0.051 g.
Spermatogenesis in this species is aestival and sperms are stored for use in the year following production.

Keywords spermatogenesis, Grass snake, Natrix natrix, northern Iran

1. Introduction

The Grass Snake, Natrix natrix (Linnaeus, 1758) is a non-
venomous colubrid snake. It is often found near water and
feeds almost exclusively on amphibians. Its subspecies
include Natrix natrix natrix, N. n. setti, N. n. corsa and
N. n. helvetica. Populations of the Eurasian mainland,
North Africa, and islands of the eastern Mediterranean
Sea are divided into eastern (N. n. natrix) and western (N.
n. helvetica) subspecies (Rastegar-Pouyani et al., 2008).
They are semi-aquatic snakes that live in moist areas
usually very close to water in the form of streams, rivers
and ponds, but they are sometimes found in dryer habitats
up to 3 km away from water (Hutchins et al., 2003).

Two spermatogenetic cycles, vernal and aestival, have
been described in temperate colubrid snakes. In both
cycles, mating occurs in spring, although vernal species
produce spermatozoa in spring just before mating, while
aestival species yield spermatozoa from the previous
summer. Aestival spermatogenesis is common in snakes
from temperate to cold regions, whereas the vernal cycle
is presented by the species inhabiting the southern belt of
the Palaearctic region (e.g., Northern Africa). The species

* Corresponding author: Dr. Vida HOJATI, form Department of Biology,
Islamic Azad University, Damghan Branch, Damghan, Iran, with her
research focusing on developmental biology.

Email: V. Hojati@Damghaniau.ac.ir

Received: 6 November 2010 Accepted: 4 February 2011

exhibiting the vernal cycle are restricted to warm regions,
where longer activity period allows the completion of
a complete reproductive cycle within a calendar year
(Feriche et al., 2008). Thus, the snakes in colder climate
with a shortened reproductive season do not possess the
vernal reproductive cycle. For this reason, we investigate
the spermatogenic cycle of N. natrix occurring in Iran
to determine which seasonal cycle (vernal or aestival) it
possesses from this locality.

2. Material and Methods

2.1 Study area The study area is located in Sari County
(54°7'E, 36°32'N) in Mazandaran, northern Iran, and on
the southern coast of the Caspian Sea. Four sampling sites
near Sari City were selected, including the Ab-Bandansar
fish training pond, Shahid Zaree Forest, Rice field and the
Tajan River, where climate is wet and temperate, with the
mean temperatures of the coldest and warmest seasons
being 1.6°C and 22.5°C, respectively. The most dominant
plants are grasses belonging to the family Gramineae.

2.2 Sampling Sampling was carried out every 15 days
from April to November, 2008 and once in 5 November
2008. Thirteen sampling trips were conducted in total.
The sampling was done at day time from 0800 to 1600
hours, and most snakes were captured around noon.
Totally, 92 specimens were captured by hand and net. The
captured specimens were identified based on the available

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literature (Latifi, 1991).

2.3 Sexual differences Females are heavier and larger
than males, especially in late spring when females
are gravid, but the tail of males is longer. Males have
noticeable swellings at the vent.

2.4 Experimental methods The specimens were
preserved in 96% ethanol. Body and tail length were
measured with a dial caliper with an accuracy of 0.02
mm. Gonads were extracted and analyzed for metric
studies. Twenty-six specimens were males, and their
testes were analyzed. The weight and volume of right and
left testes were measured separately in different times.
For studying the stages of spermatogenesis we prepared
the cross sections of testes. After fixation the testes in
Boin’s fixative, dehydratation, clearing, infiltration,
embedding, trimming, section, hydration, staining (H&E)
and mounting were done. The sections were studied by
light microscopy with 400x magnificent. Photographs
were prepared by digital camera.

3. Results

3.1 Seasonal activity The main food source for this
snake is amphibians occurring in this area (Rana
ridibunda and Bufo viridis), and includes fish. Young
snakes mainly feed on large water insects, but more
commonly on tadpoles and small fish. After breeding
in the summer, this snake becomes more active, and is
found moving several hundred meters in a day. Prey items
tend to be large compared to the size of the snake. The
snakes which have recently eaten move less and usually
stay there for a relatively long time, basking to optimize
their body temperature until the prey item is digested.
A snake may only need two or three large prey items
throughout an entire season. Fat stores in the body during
non-reproductive season and is metabolized during the
reproductive season. In this study the mean length of
body was 636.21 mm. Reproduction occurs once a year.
As spring approaches, the males emerge first and spend
much of the day time basking in an effort to raise body
temperature and thereby metabolism. This may be a tactic
to maximize sperm production as the males mate with the
females. Figure 1 shows the Grass snake in Iran.

3.2 Hemipenes In males, one pair of white or light pink
hemipenes are located at the base of the cloaca. Their
length is about 5-7 mm with 2.0-2.5 mm in diameter.
They have 3-5 large spicules with many tiny filaments
which help in the mating process. Figure 2 shows the
hemipenes of N. natrix.

3.3 Testes Testes are elliptical, white, with many blood
vessels on their surface and located in the posterior one
third of the body. The right testis is positioned more

Figure 1 The Grass snake, N. natrix in Iran (Faghiri, 2010)

Figure 2 The hemipenes of the Grass snake, N. natrix (Faghiri,
2010)

cranially than the left one. The testes are similar to those
of other reptiles in consisting of a mass of seminiferous
tubules surrounded by a connective tissue tunica
albuginea. Thin connective tissue septa from the tunica
are found between the tubules. The mean volume of testes
is 524.54 mm’° and 569.06 mm’ on the right and left,
respectively, with the mean weight being 0.051 g. The
mean diameter of seminiferous tubules is 0.17 mm. The
mean weight and volume of the testes in different months
are shown in Figures 3 and 4. Also, the minimum weight
and volume of testes were observed in late July.

3.4 Spermatogenesis The recent examination of spring
animals after hibernation revealed completely regressed
testes. Seminiferous tubules are closed in early April
and usually open in late April. Only spermatogonia and
Sertoli cells are present in April and May. No sperms
are observed in the lumina of the tubules at this time. It
seems that the regression of these structures continues
until early June. Primary and secondary spermatocytes
and undifferentiated spermatids are present in June.
Spermatids are seen in late June. Large numbers of
sperms are present in the ductus deferens throughout

No. 1

Weight of left testis (g)

12 3 4 5 67 8 9

Sampling time

10 11 12 13

Afshin FAGHIRI et al. The Spermatogenic Cycle of Natrix natrix 57

re Weight of right testis (g)

0.5
0.4
0.3
0.2
0.1

0

12 3 4 5 6 7 8 9 10

Sampling time

11 12 13

Figure 3 The mean weight of the right and left testes of N. natrix from April to November. The numbers 1 to 12 show the times of sampling

from April to September, twice in each month; and the 13 shows the single sample in November. The number of samples at each time point

was: N1=0; N2=1; N3=2; N4=2; N5=0; N6=4; N7=4; N8=5; N9=2; N10=3; N11=2; N12=0; N13=1.

Volume of left testis (mm*)

12 3 4 5
Sampling time

6 7 8 9 10 It 12 13

Volume of right testis (mm*)

Ml illli

9 10 11 12 13
Sampling time

600
500
40
30
20
10

O booo

Figure 4 The mean volumes of right and left testes from April to November

spring into the beginning of spermiogenesis in late
summer. In July, seminiferous epithelial heights and
tubule diameters increase. Spermatids become abundant
during late July. Early spermiogenesis begins in late July
and accelerates in this period. Spermiogenesis continues
from August to October. Tubule diameters and epithelial
heights reach maximum sizes during this period. By
late September germinal epithelium has been reduced.
Spermatids contain the most numerous cells with the
outer layer consisting of spermatogonia, Sertoli cells,
and occasional primary spermatocytes. Even though
males are in breeding condition at this time, no evidence
of mating was reported. Early regression begins in late
October and early November with germinal epithelium
exhausted and reduced to one to three layers. Interstitial
cells occur at all months. They are situated in connective
tissue between the somniferous tubules containing
numerous capillaries. Sertoli cells have generally one or
two nucleoli and are restricted to the outer one or two
rows of cells during spermiogenesis. In non-reproductive
seasons, spermatogonia, spermatocytes and spermatids
are present in the tubules, but mature sperms are just
observed in the reproductive season. Figure 5 shows
the stages of spermatogenesis in different months from
April to November. The testicular histology shows that
spermatogenesis begins in early June, reaches its peak
in late August and early September, and ends in early

November. These results confirm that spermatogenesis of
N. natrix of northern Iran is aestival.

4. Discussion

Studies on the reproductive biology of individual species
provide data necessary for testing hypotheses that seek
to explain the diversity of life histories observed in
squamate reptiles. Studying the reproductive cycle in
different parts of a snake’s range allows us to see the
extent of geographic variation in reproduction within
a species or family. Of the climatic factors likely to
influence reproduction, only temperatures low enough to
require prolonged hibernation play an important role in
the determination of the reproductive cycle of the males.
In regions where there is no prolonged hibernation,
male sexual activity may occur at any time of the year,
depending on the reproductive cycle of the females.
However, it is always dependent upon the climate.
Storage of sperm in the vas deferens of males may be
indispensable in the reproductive cycles of aestival type,
but prolonged retention of sperm in the oviducts, while
permitting fertilization in the absence of males, plays only
a very minor role in the harmonization of the reproductive
cycles of both sexes in the snakes (Saint Girons, 1982).
Previous studies have indicated that the specimens are
about 600 mm long when they reach sexual maturity

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Figure 5 Seminiferous tubules of Natrix natrix. Spermatogonia
and Sertoli cells are present on 20 April (A), and spermatogonia
and Sertoli cells present on 5 (B) and 20 (C) May; primary and
secondary spermatocytes and spermatids present on 20 June (D);
seminiferous epithelial heights and tubule diameters increase on
5 (E) and 20 (F) July, and spermatids become abundant and early
spermiogenesis begins during late July; mature sperms present
in lumen on 5 (G) and 20 (H) August; mature sperms present in
lumen on 5 September (I); and testes regression with germinal
epithelium reduces on 5 November (J). All slides are shown at 400x
magnificence.

(Areste and Cebrian, 2003). Although, this result probably
could be different depending on the geographic altitudes.
In this research, the mean total length of the mature
snakes was 636.21 mm. Seasonal testicular histology
of two species of the family Colubridae, Masticophis
taeniatus and Pituophis melanoleucus was described
(Goldberg and Parker, 1975). It seems likely that the
overwinter storage of sperms for as long as five months
in the vas deferens would require some form of nutrition.
Epithelial cells lining this organ are in touch with sperm
heads and this association supplies nutritional benefits.
The spermatogenic cycle of N. natrix in northern Iran
resembles that of the other snakes of the family in which

spermatogenesis occurs during late summer with sperms
stored in the vas deferens through winter and mating
in spring. The presence of sperms in the vas deferens
in those months indicates that these snakes possess the
potential for breeding throughout the year although it is
apparently restricted to spring and early summer. The
reproduction of other species of Colubridae has been
studied by Goldberg. In the long-nose snake, Rhinocheilus
lecontei, males follow a seasonal testicular cycle with
sperm produced from July to October; regressed testes
were present from March to July. Males with recrudescent
testes were present from April to August. Breeding
presumably occurs in spring although sperm was present
in the vas deferens during all months examined (Goldberg,
2000). In the night snake, Hypsiglena torquata, males
follow a seasonal testicular cycle with sperm produced
from April to September. The prolonged period of sperm
production suggests a similarity to testicular cycles of
some tropical snakes (Goldberg, 2001). In the Chihuahuan
hook-nosed snake, Gyalopion canum, males follow a
seasonal testicular cycle with sperm produced from June
to September. Testicular recrudescence appears to occur
in the spring (Goldberg, 2002). The viperine snake,
Natrix maura (Colubridae) emerged from hibernation
in March and mating occurred from April to June, using
the sperms stored by males overwinter. Spermatogenesis
was postnuptial and sperm mobilization from testes to
spermiducts occurred in August (Santos et al., 2000). In
the lined snake, Tropidoclonion lineatum (Colubridae),
sperms are stored in the vas deferens throughout winter
and early spring, indicating a possibility of spring mating.
By mid-June, sperms are absent from the vas deferens.
The diameter of the tubules within the sexual segment of
the kidney increases significantly from May to September
with a reduction in tubule diameter by late September.
All male lined snakes are capable of mating at one year
of age (Randolph and Aldridge, 1985). Spermiogenesis
is a conservative mechanism in squamates, with few
differences existing among the studied species. One of
these variations includes lipid droplets in the nuclear
region of the late spermatids in snakes (Hamilton and
Fawcett, 1968). Recently the ultrestructure of mature
sperm has been used in phylogenetic analyses (Jamieson,
1995). Our results show that in northern Iran, with
temperate climate, the spermatogenic cycle of N. natrix is
aestival.

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