The effect of different extenders on the motility and

Research Article
Turk. J. Vet. Anim. Sci.
2012; 36(2): 177-182
The effect of different extenders on the motility and
morphology of ram sperm frozen or stored at 4 °C
Recai KULAKSIZ1, Çiğdem ÇEBİ2,*, Ergun AKÇAY2
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Kafkas,
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Ankara,
Ankara - TURKEY
Received: 09.03.2011
Abstract: This study was conducted to evaluate the effects on sperm motility and abnormality in ram semen extended
with skimmed milk (M), sodium citrate (SC), Tris (T), and Bioxcell® (B) after storage in liquid form at 4 °C and in frozen
form. Ejaculates were collected from 3 Akkaraman rams by artificial vagina twice a week during the non-breeding
season. After pooling, each pooled ejaculate was split into 4 equal aliquots and diluted with skimmed milk (M), sodium
citrate (SC), Tris (T), and Bioxcell® (B) extenders. Sperm motility was significantly higher (P < 0.05) in M compared
with B, SC, and T on the first and second days (48 h) of storage. With regard to the percentage of total abnormal
spermatozoa, the results in M were different from (P < 0.05) from those of SC and T on the first day (24 h) of storage but
no different from B. Differences among extenders were found to be significant post-thawing for spermatozoa motility
and the percentage of total abnormal spermatozoa. The total abnormality of semen diluted with M was significantly
lower than that observed in the other extenders. Consequently, it was found that skimmed milk was better than the
other extenders in terms of the sperm parameters evaluated during liquid storage and post-thaw.
Key words: Ram semen, extenders, liquid storage, cryopreservation, sperm parameters
Koç spermasının kısa süreli saklanması ve dondurulmasında farklı sulandırıcıların
motilite ve morfoloji üzerine etkisi
Özet: Bu çalışma, koç spermasının yağsız süt tozu (M), sodyum sitrat (SC), Tris (T) ve Bioxcell® (B) sulandırıcıları ile
sulandırılıp, +4 °C de kısa süreli saklanması ve dondurulması sonrası spermatozoa motilitesi ve anormalitesi üzerine
etkisinin saptanması amacıyla yapıldı. Ejakulatlar 3 Akkaraman koçundan sezon dışı dönemde suni vajen yardımıyla
haftada 2 kez alındı. Alınan ejakulatlar birleştirildikten sonra 4 eşit hacme bölünmüş ve yağsız süt tozu (M), sodyum
sitrat (SC), Tris (T) ve Bioxcell® (B) sulandırıcıları ile sulandırılmıştır. Kısa süreli saklamanın 24 ve 48. saatlerinde elde
edilen en yüksek spermatozoa motilitesi yağsız süt tozunda elde edildi. Saklamanın 24. saatinde, anormal spermatozoa
oranı bakımından, yağsız süt tozu sulandırıcısı ile Tris ve sodyum sitrat sulandırıcı içeren gruplar karşılaştırıldığında
istatistiksel olarak aradaki farklılıklar önemli bulunurken (P < 0,05), Bioxcell® ile yağsız süt tozu arasındaki farklılıklar
istatistiksel açıdan önemsiz bulunmuştur. Çözdürme sonrası spermatozoa motilitesi ve total anormal spermatozoa oranı
üzerine sulandırıcılar arasındaki farklılıklar önemli bulunmuştur. Süt tozu sulandırıcısında, total anormal spermatozoa
oranı diğer sulandırıcılara göre önemli derecede daha düşüktür. Sonuç olarak, çözdürme ve kısa süreli saklama sonrası
bazı spermatolojik parametreleri değerlendirildiğinde yağsız süt tozu sulandırıcısının diğer sulandırıcılardan daha iyi
sonuçlar verdiği saptandı.
Anahtar sözcükler: Koç sperması, sulandırıcılar, kısa süreli saklama, kriyopreservasyon, sperm parametreleri
* E-mail:
The effect of different extenders on the motility and morphology of ram sperm frozen or stored at 4 °C
Akkaraman is a fat-tailed, indigenous breed
constituting 45% of the sheep population in Turkey.
Most lamb meat production is from this breed
(1). However, this breed generally produces low
yields. Therefore, studies on crossing Akkaraman
sheep with high-producing sheep breeds have been
widely used to improve their yield traits. Artificial
insemination (AI) allows for the rapid dissemination
of genetic material from a small number of superior
sires to a large number of females (2). Semen from
farm animals used for this purpose can be stored in
liquid form at 4 °C for a short time or it may be kept
for a long period in a cryopreserved state with liquid
nitrogen (3).
For the effective use of artificial insemination
techniques in the sheep industry, investigation on
the methods of ram semen dilution and freezing
is necessary. Many extenders have been used for
freezing ram semen. Semen has been usually diluted
with Tris plus egg yolk, glucose phosphate solution,
egg yolk-citrate solution, homogenized whole milk,
fresh and dried skim milk, lactose solution, and
commercial diluents (3-5).
The successful storage of ram semen in liquid
and frozen forms depends on the composition of
the extender used. The recommended maximum
storage time without impaired fertility after AI
has traditionally been said to be as short as 6-12
h. Storage diluents and techniques have been
developed and adapted with the aim of improving
the cryopreservation of ram semen, but fertility
after cervical AI with frozen-thawed sperm remains
significantly lower than that achieved with fresh
semen. Some factors that contribute to the reduced
viability of cryopreserved ram sperm include
extender composition, cryoprotectant concentration,
egg yolk from different avian species, and the
cooling, freezing, and thawing rates, as well as the
quality of semen used. Different cryoprotectants or
extenders have been used to prevent cryoinjuries.
The most common cryoprotectant in use for sperm
cryopreservation is glycerol. A suitable diluent is
a basic necessity for the successful preservation of
spermatozoa and for obtaining higher conception
rates in field trials using diluted semen (5-8).
Although information is available on stored
ram liquid semen up to 24 h, there is a need to
further investigate the short-term and long-term
preservability of Akkaraman ram semen for the
extensive utilization of this valuable germplasm by
artificial insemination.
This study was designed to evaluate and compare
the quality (subjective motility and total abnormality)
of Akkaraman ram spermatozoa preserved at 4 °C
for up to 48 h and at freezing temperatures using 4
different extenders.
Material and methods
Animals and semen collection
The rams were housed at the Education Research and
Practice Farm in the Faculty of Veterinary Medicine
at the University of Ankara. The rams were kept
under natural light and maintained under a uniform
and constant nutritional regime, with each ram being
fed a daily diet of 1 kg of concentrate, dried grass, salt
lick, and water ad libitum.
Using an artificial vagina, semen samples were
obtained from 3 mature Akkaraman rams (3 and 4
years of age) with proven fertility. A total number of
21 ejaculates were collected from the animals during
the non-breeding season (summer). Semen samples
were pooled to eliminate individual differences. From
this, 7 pooled ejaculates were included in the study.
Semen evaluation
The ejaculates were evaluated and accepted if the
following criteria were met: a volume of 0.75 mL, a
sperm concentration >2.5 × 109 spermatozoa/mL;
sperm motility >70%, and a total morphological
abnormality frequency of <20.
Sperm motility was assessed using a phase contrast
microscope (×400 magnification), with a warm stage
maintained at 37 °C. A wet semen mount was made
using 2 μL of semen placed directly on a microscope
slide and covered by a cover slip. For each sample, at
least 5 microscopic fields were examined by 2 trained
observers. The mean of the 2 successive evaluations
was recorded as the final motility score (9).
For the sperm morphology assessment, a small
drop of semen was added to Eppendorf tubes
containing 0.5 mL of Hancock’s solution (10). A
single drop of this mixture was put on a microscope
slide and covered with a cover slip. The percentage
of abnormal sperm (detached heads, acrosomal
aberrations, abnormal mid-pieces, or tail defects) was
recorded by counting a total of 200 spermatozoa under
phase contrast microscopy (×1000 magnification; oil
Semen extenders
We used 4 extenders (T, SC, M, and B) in the present
study. As a first step, the extenders, without gylcerol,
were used for short term storage and prepared as
Extender T
Tris-citric acid egg yolk extender was prepared by
using 3.63 g of tris-(hydroxymethyl)-aminomethane,
1.99 g of citric acid, 0.5 g of glucose, and 20% egg yolk
in 80 mL of distilled water.
Extender SC
Sodium citrate extender was prepared from a 2.9%
aqueous solution of trisodium citrate and 20% egg
yolk in 80 mL of distilled water.
Extender M
Milk extender was prepared by using 10 g of
skimmed milk powder and 0.9 g of glucose in 100
mL of distilled water, heated to 95 °C for 10 min, and
then cooled to room temperature before the addition
of 10% egg yolk (6).
Semen freezing
The pooled semen was diluted with 4 different
extenders, namely the T, SC, and M extenders, which
included egg yolk, and the egg yolk-free diluent B.
Each pooled ejaculate was split into 4 equal aliquots
and diluted with the 4 different extenders for a total
of 4 experimental semen groups (37 °C) at a final
concentration of 800 × 106 spermatozoa per milliliter.
Following dilution, the sperm was drawn into 0.25
mL plastic straws (IMV, Laigle, F-61300, France)
and sealed with polyvinyl alcohol (PVA). Straws
were equilibrated at 4 °C for 2 h and spermatozoa
motility and total abnormal spermatozoa rates
were assessed at the end of this period to determine
differences in extenders in a water bath at 37 °C for
30 s for microscopic evaluation. After equilibration,
the straws were suspended on a styrofoam rack 4 cm
above the liquid nitrogen (vapor) for 15 min. The
straws were then plunged into the liquid nitrogen,
where they were stored until thawing. After storage
for a period of 4 weeks, the semen straws were thawed
in a water bath (37 °C for 30 s) for microscopic semen
evaluation immediately after thawing.
Statistical analysis
Experiments were replicated at least 7 times. Data
were expressed as the mean ± standard error of the
mean (SEM). Data were subjected to analysis of
variance (ANOVA) using mixed-model procedures.
All analyses were carried out using the SPSS 12 for
Extender B
The Bioxcell® extender was prepared according
to the manufacturer’s (IMV technologies, L’Aigle,
France) instructions. Bioxcell® (egg-yolk free and
concentrated medium) is a commercial diluent.
In the second step, 7% glycerol was added to
each extender (with the exception of Bioxcell®) for
cryopreservation of the ram semen.
Semen preservation
Short-term preservation
Each ejaculate was equally transferred into 4 tubes and
diluted with extenders at 1:10 (v/v) rates. The daily
spermatozoa motility and abnormal spermatozoa
rates during storage at 4 °C were determined in each
of the different extenders.
The data on motility and abnormal spermatozoa
preserved in T, SC, M, and B extenders for 2 days
at 4 °C are presented in Table 1. Spermatozoa
motility was significantly higher (P < 0.05) in M
(68.5, 47.5%) compared with B (55, 40%), SC (51.2,
25.5%), and T (48.7, 36.5%) at the first and second
days of storage. The percentage of total abnormal
spermatozoa in M (18.7%) was different from (P <
0.05) that of SC (27.3%) and T (26.5%) on the first
day of storage but the rate was not different from that
of B (23.8%). On the second day of storage, however,
the total abnormality was not found to be different
in any of the extenders. The percentage of abnormal
spermatozoa increased with the number of days in
storage for all of the extenders.
11.0 ± 0.2a
9.5 ± 0.3a
26.5 ± 0.4a 18.7 ± 0.2b
B: Bioxcell®
SC: Sodium citrate
M: Skimmed milk
T: Tris
*Groups with different letters (a, b) in the same row are significantly different (P < 0.05).
8.5 ± 0.2a
10.2 ± 0.3a
abnormality (%)
27.3 ± 0.3a
51.2 ± 0.5a
24 h
82.5 ± 0.1a 84.0 ± 0.1a 80.0 ± 0.2a 84.5 ± 0.1a 48.7 ± 0.3a 68.5 ± 0.3b
Motility (%)
36.5 ± 0.2a 47.5 ± 0.4b 25.5 ± 0.5a 40.0 ± 0.4a
23.8 ± 0.2ab 37.3 ± 0.3a 45.5 ± 0.6a 45.1 ± 0.5a 37.5 ± 0.7a
55.0 ± 0.6a
48 h
Table1. The mean percentage of motility and total abnormality of ram spermatozoa preserved for 2 days at 4 °C in 4 different extenders.
The effect of different extenders on the motility and morphology of ram sperm frozen or stored at 4 °C
Differences among the extenders in terms of
spermatozoa motility and total abnormality were
found to be significant post-thawing. The data on
motility and abnormality post-thawing in the T,
SC, M, and B extenders are presented in Table 2.
Spermatoza motility was significantly higher (P
< 0.05) in M (21.2%) compared with B (7.0%), SC
(11.5%), and T (16.2%) after being thawed. The total
abnormality of semen diluted in M (50%) was lower
than that observed in the other extenders.
Table 2. The mean percentage of motility and total abnormality
of ram spermatozoa after freeze-thawing in 4 different
Total abnormality
16.2 ± 0.2a
55.4 ± 0.5ab
21.2 ± 0.1b
50.6 ± 0.8a
7.0 ± 0.1c
65.0 ± 0.5c
11.5 ± 0.2c
60.0 ± 0.4bc
Groups with different letters (a, b, c) in the same column are
significantly different (P < 0.05).
T: Tris
M: Skimmed milk
B: Bioxcell®
SC: Sodium citrate
however, Paulenz et al. (15) and Gündoğan (16)
reported better spermatozoa motility and membrane
integrity rates in a Tris-based extender than in the
sodium citrate and skimmed milk extenders. Lopez
et al. (17) observed no differences between sodium
citrate-, Tris-, and milk-based extenders when
subjected to liquid storage at 4 °C. The post-thawing
sperm motility (16.2%) determined for the Tris
extender used in our study was close to the findings
of Tekin et al. (18) but Soylu et al. (19) reported better
spermatozoa motility and membrane integrity in Tris
extender than that found in our study.
To our knowledge, no previous study is available
on the liquid preservation of ram semen in prepared
extenders containing soy lecithin (Bioxcell) as an egg
yolk substitute. In the present study, sperm motility
showed higher values for Bioxcell compared to Tris
and sodium citrate extenders as the liquid of storage.
However, the parameters used for semen quality
in this study showed significantly lower values for
Bioxcell post-thawing in comparison with the other
extenders. However, Gil et al. (20) found that Bioxcell
was not different from a skimmed milk extender for
the freezing of ram semen.
From the present results it was observed that the type
of extender used in diluting Akkaraman ram semen
is an important factor in the successful preservation
(at 4 °C in the refrigerator) and cryopreservation (at
−196 °C in liquid nitrogen) of ram spermatozoa.
It is most likely that the cryogenic damages are due
to the irreversible destruction of single components
of the structural organization of sperm cells. Sperm
are subjected to major changes in osmotic pressure
during freezing and thawing. The stress on sperm
membranes is dependent upon the basic extender
used and the concentration of cryoprotectant as
they interact with the freezing and thawing rates
(21-23). Moreover, in the present study, the portion
of ejaculates collected during a non-mating season
(summer) had lower motility values and a higher
percentage of total abnormal spermatozoa in all of
the extenders after freezing.
Many extenders have been used for freezing ram
semen and reports show that the type of extender
used plays an important role in the successful
preservation of ram semen (11-13). Skimmed milk
extenders preserve sperm motility better than other
extenders. These findings were in agreement with
the results of Lopez-Saez et al. (13) and Kulaksız
et al. (14) on ram semen. Contrary to our results,
In conclusion, the freezability of Akkaraman ram
semen was adversely affected by the summer term
in which the samples were collected. The skimmed
milk extender showed the best liquid storage and
post-thaw motility compared to the other extenders.
Regardless of the extender type, however, semen
freezing increased the rate of sperm morphological
The effect of different extenders on the motility and morphology of ram sperm frozen or stored at 4 °C
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