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R. KULAKSIZ, Ç. ÇEBİ, E. AKÇAY

179

single drop of this mixture was put on a microscope

slide and covered with a cover slip.  e 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 magni cation; oil

immersion).

Semen extenders

We used 4 extenders (T, SC, M, and B) in the present

study. As a  rst step, the extenders, without gylcerol,

were used for short term storage and prepared as

follows:

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).

Extender B

 e 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.  e daily

spermatozoa motility and abnormal spermatozoa

rates during storage at 4 °C were determined in each

of the di erent extenders.

Semen freezing

 e pooled semen was diluted with 4 di erent

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 di erent extenders for a total

of 4 experimental semen groups (37 °C) at a  nal

concentration of 800 × 10

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

di erences in extenders in a water bath at 37 °C for

30 s for microscopic evaluation. A er equilibration,

the straws were suspended on a styrofoam rack 4 cm

above the liquid nitrogen (vapor) for 15 min.  e

straws were then plunged into the liquid nitrogen,

where they were stored until thawing. A er 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 a er 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

Windows.

Results

 e 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 signi cantly 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  rst and second

days of storage.  e percentage of total abnormal

spermatozoa in M (18.7%) was di erent from (P <

0.05) that of SC (27.3%) and T (26.5%) on the  rst

day of storage but the rate was not di erent from that

of B (23.8%). On the second day of storage, however,

the total abnormality was not found to be di erent

in any of the extenders.  e percentage of abnormal

spermatozoa increased with the number of days in

storage for all of the extenders.

1 2 3 4 5 6

Research Article 1
References 6

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