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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. 

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 

56 Asian Herpetological Research 

Vol. 2 

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, 

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) 



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 


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 

58 Asian Herpetological Research 

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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 

(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 


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