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Motility and Viability of Friesian Holstein Spermatozoa in Three Different

Extenders Stored at 5ºC

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 MOTILITY AND VIABILITY OF FRIESIAN HOLSTEIN SPERMATOZOA
IN THREE DIFFERENT EXTENDER STORED AT 5oC

R. I. Arifiantini and B. Purwantara

Department of Veterinary Clinic, Reproduction and Pathology,
Faculty of Veterinary Medicine,Bogor Agricultural University,
Darmaga Campus, Bogor 16680 - Indonesia
Corresponding E-mail: [email protected]

Received October 6, 2010; Accepted November 18, 2010

ABSTRACT

The aims of this study was to compare Tris egg yolk and Citrate egg yolk extender and
supplementation of fructose on citrate egg yolk on the quality of Friesian Holstein (FH) bull semen
stored at 5 oC. Semen was collected from 5 FH bulls using an artificial vagina. The semen were
evaluated macroscopic and microscopically. The semen divided into three tubes and extended with Tris
egg yolk (TEY), Citrate egg yolk (CEY) or Citrate fructose egg yolk (CFEY). Extended semen was
stored at 5 oC and evaluate daily for sperm motility and viability. There was no significant differences
(P>0.05) on the sperm viability among three extender, for every time observation during 144 hours of
storage. This similar finding found on the sperm motility in all extender for 48 hours of storage. The
sperm motility in TEY demonstrated significantly greater (P<0.05) than in CFEY and CEY extender at
72 to 120 hours storage. In the end of storage, sperm motility in TEY (35.2 ± 4.1%) and CFEY (33.5 ±
2.71%) extender statistically indicated no significant different, and both were greater than CEY. In
conclusion, CFEY support the sperm motility as good as TEY of FH bull.
Keywords: Citrate, Fructose, FH semen, Tris, citrate

INTRODUCTION semen preservation in bull (Verberckmoes et al.,

2005; Arifiantini et al., 2005;2006; 2010), ram
The use of frozen-thawed semen facilitates (Paulanz et al., 2003; Purdy et al., 2006; Nel-
distribution of semen world wide and enables Themaat et al., 2006; Soylu et al., 2007), buffalo
screening of bulls for infectious diseases before (Rasul et al., 2000; Sukhato et al., 2001), buck
their semen is used in the field. In rare cases when (Fukui et al., 2007; ), canine (Schafer-Somi et al.
liquid nitrogen is limited, chilled semen is an 2006; Hermansson and Linde-Forsberg, 2006) and
alternative used for AI. Various organic salt and elephant (Graham et al., 2004), and Spanish ibex
acid combinations have been tried for semen (Santiago-Moreno et al., 2006).
preservation, along with proprietary biological Fructose known as a simple sugar and has
buffers. The general requirements for semen been used in bull and ram (Vishwanath and
diluents are: ionic or non-ionic substances to Shannon, 2000), canine (Hermansson and Linde-
maintain the osmolarity and to buffer the medium; Forsberg, 2006; Yilziz et al., 2000), stallion
a source of lipoprotein or high molecular weight (Arifiantini et al., 2009) and buck (Naing et al.,
material to prevent cold shock, such as egg yolk 2010) semen preservation. The aims of this study
or milk; glucose or fructose as an energy source; was to compare Tris egg yolk and Citrate egg yolk
and other additives such as enzymes and extender and supplementation of fructose on
antibiotics (Vishwanath and Shannon, 2000). citrate egg yolk on the quality of FH bull semen
In years of 1941 the short-term storage storage at 5oC.
extender for bull spermatozoa consisted of 2.9%
sodium citrate and egg yolk, later in year of 1960, MATERIALS AND METHODS
Tris-based extender was shown to be superior to
sodium citrate for bull semen preservation A total of five FH bulls belong to Lembang
presumably due to its better buffering capacity. Artificial Insemination Centre were used as sperm
Since then Tris-based extender widely use for

222 J.Indonesian Trop.Anim.Agric. 35(4) December 2010

donors. Semen was collected by using an artificial The percentage of motile and viable sperm
vagina once a week for 4 weeks. The semen was evaluated every 24 hour, for 144 hours
samples were assessed for volume, color, storage. The motile sperm evaluate by mixing the
consistency, mass activity, sperm concentration, semen gently and placing a 10 µL drop of diluted
sperm morphology, sperm viability and semen on a warm slide and covered with a glass
percentage of motile sperm. Only ejaculates with cover slip (18x18mm) from five selected
a concentration of greater than 800×106 representative fields. The mean of the five
sperm/mL, having >70% progressively motile estimations was recorded as final motility score.
sperm and >80% of the sperm with normal Sperm viability was assessed using nigrosin–eosin
morphology were selected for this study. A total stain (Bart and Oko, 1989). Placing 10 µL drop of
of 20 ejaculates were individually process for diluted semen on a slide and adds with 40 µl drop
preservation of nigrosin–eosin, and smears on a slide and
drying quickly in heating stage (37oC).
Media Preparation Microscopes were selected randomly from ten
All chemicals were obtained from Merck, fields, with total of 200 cells. Individual sperm
Germany. The cooling extender consisted of three were recorded as being viable (unstained) or dead
buffer: Tris, Citrate and Citrate fructose (Table 1). (stained).
All buffer added with 20% (v/v) egg yolk and
antibiotics (500 IU penicillin and 500µg Data Analysis
streptomycin per mL). A total of three extender The data were statisticall analyzed for
were prepared i.e. Tris egg yolk (TEY), Citrate differences among the means by one way analysis
egg yolk (CEY) and Citrate fructose egg yolk of variance. The Turkeys test was use to compare
(CFEY). treatment means using statistical soft ware
Minitab 14 version.
Semen Evaluation and Processing
The macroscopic evaluations included semen RESULTS AND DISCUSSION
volume, pH, consistency and color. The
microscopic evaluation conducted under Raw semen demonstrated a variation on the
microscope 100-400x magnification (Olympus quality between the bulls. The mean of semen
CH 20) included mass activity and percentage of volume was 6.46±1.26 mL, cloudy to creamy
progressive motility [0 (not motile) –100% (100% white in color, pH was 6.21±0.07, and thin to
motile], velocity [1 (very slow)-5 (very fast)], thick in consistency. The mean of mass activity
viable sperms using eosin-negrosin (Barth and was 2.28 ± 0.41 with the percentage of motile and
Oko 1989), sperm concentration using Neubauer viable sperm were 72.5±2.3% and 89.29±1.51%
counting chamber (Kirkman-Brown and respectively. The individual scoring was
Björndahl, 2009) and sperm morphology was 3.69±0.53 and 1032.00±232.06 x106/mL in sperm
assess with carbolfuchsin-eosin (Al-Makhzoomi concentration.
et al. 2008). After evaluation, each of raw semen The sperm motility and viability
was equally divided into three tubes, and diluted demonstrated a gradual decrease during 144 hour
in one of three extender; TEY, CEY and CFEY to storage, which was correlated with the storage
reach the total semen concentration of 30x10 6/ time (Table 2 and Table 3). The motile sperm
mL. Extended semen then stored at 5oC for daily decrease between 4.3 to 8.6% for every 24 hours
evaluation. observation and viable sperm decrease between

Table 1. Buffer Composition

Composition Tris Citrate Citrate fruc tose

Tris-hydroxymethyl-aminomethane (g) 3.03 2.9 0
Citric ac id monohydrate (g) 1.78 0 0
Fruc tose (g) 1.25 0 1.25
Sodium citrate dehydrate (g) 0 0 2.32
Aquadest ad (mL) 100 100 100

Friesian Holstein Semen Preservation (R.I. Arifiantini and B. Purwantara) 223

5.1 to 5.8% (data not show). Sperm require (P>0.05) on the percentage of sperm motility on
nutrients to maintain their cellular activity; the those extended in CFEY and CEY at 96 as well as
main source of nutrients is simple carbohydrate, at 120 hours of storage. In the end of storage
such as fructose or glucose, in seminal plasma or sperm motility in TEY (35.2 ± 4.1%) and CFEY
diluents content (Vishwanath and Shannon, 2000). (33.5±2.71%) extender statistically indicated no
Carbohydrates were broken down through significant different, both were greater than CEY
glycolysis or fructolysis to produce ATP and ADP, (Table 2).
which are the energy source of sperm; this occurs The effectiveness of TEY compare to CEY
in the mitochondria located in the sperm’s tail to and CFEY on maintained sperm motility in
midpiece. Sperm movement stops when no predictable. Several reports have revealed
carbohydrate left in semen diluents. information on Tris egg yolk as an exelent
This study demonstrated no significant extender for semen preservation in cattle bull,
differences (P>0.05) on the viability among three ram, buffalo bull (Purdy et al., 2006; Nel-
extenders, for every time observation during 144 Themaat et al., 2006; Soylu et al., 2007; Rasul et
h of storage (Table 3). The sperm viability al., 2000; Sukhato et al., 2001; Fukui et al., 2007)
associate with the intactness of sperm membrane. and canine (Schafer-Somi et al., 2006;
The addition of egg yolk which contains Hermansson and Linde-Forsberg, 2006).
phospholipids and lecithin (Bergeron et al., 2004) In this study it was found that the addition of
in all extender might therefore protect the sperm fructose to Citrate egg yolk extender improves in
membrane against cold shock (Paulanz et al., sperm totality than Citrate egg yolk alone.
2003). When the sperm plasma membrane Fructose and glucose have been known as simple
damage is located at midpiece, aspartat sugar having low molecular weight. Low
aminotransferase enzyme (AspAT), which is the molecular weight molecules can pass through the
main mitochondrial enzyme in ATP production, is plasma membrane of spermatozoa and provide
released from the cell, and enters the seminal energy to function in metabolism and normal
plasma. Loss of AspAT interrupts ATP production physiological manner (Naing et al., 2010).
and disturbs sperm motility (Colenbrander et al., Results of this study indicated that
1992). spermatozoa stored either in TEY or in CFEY
There was no significant on the sperm diluents have the same sperm motility after 144
motility in all extender for 48 hours of storage; days of storage. This fact proved that CFEY could
the average of sperm motility was 59.5±4.4% to be use as an alternatif extender for bull semen
62.3±2.7%. At 72 until 120 hours of storage, the preservation.
sperm motility in TEY demonstrate significantly Indonesia lately has 17 AI centre and as far
higher (P<0.05) than in those diluted in CFEY or Skim milk, Tris egg yolk or commercial extender
CEY extender. We also found no significant were chosen as an extender for bull semen

Table 2. The Sperm Motility of FH Bull Semen Extended in TEY (Tris

Eggyolk), CEY (Citrate Eggyolk) and CFEY (Citrate Fructose Eggyolk)
Extender Stored at 5 oC for 144 Hours

Type of Extender
Storage time
TEY CEY CFEY
0 72.50 ± 2.3 Aa 72.50 ± 2.30 Aa 72.50 ± 2.34 Aa
24 69.00 ± 2.2 Bb 68.00 ± 2.10 Bb 67.75 ± 2.40 Bb
48 62.30 ± 2.7 Cc 59.50 ± 4.40 Cc 61.25 ± 1.98 Cc
72 55.00 ± 1.3 Dd 49.30 ± 3.00 Df 53.00 ± 1.68 De
Eg Ehi
96 47.00 ± 3.6 42.00 ± 3.40 44.25 ± 4.81 Egh
120 42.70 ± 3.2 Fj 35.80 ± 2.60 Fkl 38.75 ± 2.93 Fjk
144 35.20 ± 4.10 Gm 30.00 ± 1.53 Gn 33.50 ± 2.71 Gm
Different cap ital sup erscrip t in the same column means significantly different (p <0.05)
Different small sup erscrip t in the same line means significantly different (p <0.05)

224 J.Indonesian Trop.Anim.Agric. 35(4) December 2010

 Table 3. The Sperm Viability of FH Bull Semen Extended in TEY (Tris Eggyolk);
CEY (Citrate Eggyolk.) and CFEY (Citrate Fructose Eggyolk) Stored at
5 oC for 144 Hours

Type of Extender
Storage time
TEY CEY CFEY

0 89.194 ± 1.009A 89.194 ± 1.009A 89.194 ± 1.009A

24 83.987 ± 1.267B 83.85 ± 1.201B 83.836 ± 1.179B

48 78.917 ± 1.423 C
77.422 ± 2.567 C
77.812 ± 0.982C

72 73.517 ± 1.248D 72.359 ± 2.353D 72.364 ± 1.256D

96 68.305 ± 1.47E 67.294 ± 1.943E 67.417 ± 1.178E

120 63.221 ± 1.61F 62.245 ± 1.964F 62.215 ± 1.161F

144 56.712 ± 1.393G 57.221 ± 1.829G 56.836 ± 0.921G

Different capital sup erscrip t in the same column means significantly different (p<0.05)

cryopreservation. On the base of this research and Arifiantini, R.I and T.L Yusuf. 2006. Keberhasilan
considering the chemical price of Citrate cost was Penggunaan Tiga Pengencer Dalam Dua
less than Tris, it was suggested to use Citrate Jenis Kemasan Pada Proses Pembekuan
fructose egg yolk as an alternative extender to Semen Sapi Frisien Holstein. Majalah Ilmiah
produce bull frozen semen. Peternakan. 9:89-93.
Arifiantini, R.I., B. Purwantara, T.L. Yusuf and D.
CONCLUSION Sajuthi. 2009. Peranan Fruktosa, Rafinosa
dan Trehalosa Pada Kriopreservasi Semen
Base on this research it was concluded that Kuda. Med. Pet. 32:155-228.
Citrate fructose egg yolk comparable with Tris Arifiantini, R.I and T.L. Yusuf. 2010. Developing
egg yolk diluent for preserving FH bull semen at of Tris Soy Milk Diluent for Friesian
5oC. Holstein Bull Frozen Semen. Hayati. Biosci.
17:91-94
ACKNOWLEDGMENTS Barth, A.D. and R.J. Oko. 1989. Abnormal
morphology of bovine spermatozoa. Iowa:
This work was supported by Pusat Studi Iowa State University Press.
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BIOTROP). Thank to Maidaswar MSi, DVM the Manjunath. 2004. Low-density lipoprotein
head of Lembang Artificial Insemination Centre fraction from hen’s egg yolk decreases the
for providing the facilities of this research, binding of the major proteins of bovine
Aminah DVM and Bondan Achmadi-Faculty of seminal plasma tosperm and prevents lipid
Veterinary Medicine for technical assistance. efflux from the sperm membrane. Biol.
Reprod. 70:708–717
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226 J.Indonesian Trop.Anim.Agric. 35(4) December 2010

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