Indian J. Anim. Res., 50 (3) 2016:310-313
AGRICULTURAL RESEARCH COMMUNICATION CENTRE
Print ISSN:0367-6722 / Online ISSN:0976-0555
www.arccjournals.com/www.ijaronline.in
Practical use of percoll density gradient centrifugation on sperm sex
determination in commercial dairy farm in Thailand
Kornkanok Promthep1, Saranya Satitmanwiwat2, Narisorn Kitiyanant1, Pisit Tantiwattanakul3,
Kanya Jirajaroenrat4, Ronachai Sitthigripong4 and Chatchawan Singhapol*
Department of Biotechnology, Faculty of Applied Science,
King Mongkut’s University of Technology North Bangkok, Pracharat1 Rd., Bangsue , Bangkok 10800 Thailand.
Received: 03-04-2015
Accepted: 31-12-2015
DOI:10.18805/ijar.8427
ABSTRACT
Our experiment demonstrated an extension of Percoll® Density Gradient Centrifugation to use as a selection tool for sperm
sexing developed for dairy farmer. We develop a simple protocol for bovine sperm sexing and used for artificial insemination
(AI) and in vitro fertilization (IVF) for embryo transfer (ET). The quality of sperm after centrifugation in each layer were
significantly different (P <0.01). There was no significant difference between percentage of live sperm after centrifugation
and fresh sperm before centrifugation (P >0.05). The phenotype and swimming capacity of sperms in each layer were
significantly different (P < 0.01). It was found that the most appropriate sperm in the layer after centrifugation and after
quinacrine staining was between 65-70% Percoll®. X-bearing spermatozoa at this layer was 60.75% and motility was
95.86±0.46%. After insemination, the pregnancy ratio in the experiment was 40% which is similar to the percentage of
normal AI. The number of female offspring in the experiment was 71.4% in selected sperm vs 50% in non-selected sperm
AI. In the In Vitro fertilization experiment, sperm from our 7 layer Percoll® gradient concentration can fertilize and produce
blastocyst embryo successfully. The simple 7 layer Percoll® gradient concentration protocol is appropriate for dairy farmer
to do as a pre-AI step to increase their female offspring in their herd.
Key words: Dairy farm, Percoll® Density Gradient Centrifugation, Sperm sexing.
INTRODUCTION
As a demand for more efficiency in production,
many high technology methods have been developed for
bovine genetic improvement in dairy industry. Several
methods have been developed for Y- and X-bearing
spermatozoa selection consisting running sperm through
different gradients or flow cytometry (Sills et al., 1998; Asit
et al., 2011). However, using advance techniques mean
higher cost for dairy farmers. For a small commercial dairy
farm in Thailand, we aim to modify a simple protocol which
suitable for dairy farmers that required only a
predetermination method for female sex offspring. Sex
selection of spermatozoa has been compromised to use as a
preliminary step before artificial insemination and embryo
transfer techniques (Mota et al., 2013). In human IVF
technique, sperm can be separated using gradient density
centrifugation, based on the weight difference of X- and
Y-sperm (Moruzzi, 1978). This method is based on a density
medium solution available in various thicknesses layers in a
conical centrifuge tube. Common media using in
centrifugation through gradient layers are albumin, Percoll®
and some microbeads suspension such as SephadexTM. In
theory, after sperm centrifugation, heavier X-sperm will
sediment in a bottom layer of a gradient where as lighter Ysperm will remain at the top layer. Thus, a simple Percoll®
gradient centrifugation has been commonly used to clean
out dead sperms in reproductive laboratory, however, using
only 2 different density layers of solution. This method has
been successfully applied in human without deleterious effect
(Kaneko et al., 1983). Moreover, this 2 layers method has
been tested in bovine and swine (Kobayashi et al., 2004;
Wolf et al., 2008). In this study, we demonstrated an
extension of Percoll® Density Gradient method to use as a
simple sperm selection tool which is affordable for dairy
farm industry. We develop a simple protocol for bovine sperm
sexing and tested with artificial insemination (AI) and in
vitro fertilization (IVF) for embryo transfer (ET). The sex
ratio in offspring after separation were examined.
MATERIALS AND METHODS
Semen source: Semen was collected by artificial vagina
method from two Holstein bulls at Pakthongchai Dairy
Research Farm, Nakhonratchasima, Thailand. These bulls
*Corresponding author’s e-mail: [email protected]. 1 Reproductive Biology and Stem Cell Research Group, Insititute of Molecular
Biosciences, Mahidol University, Nakhon Pathom, 73170 Thailand, 2Reproductive Research Group, Institute of Molecular Biosciences, Mahidol
Univeristy Salaya, Nakhonpathom 73170, Thailand, 3Nexgen genetic center, Athenee Tower, Witthayu road, Lumpini Pathumwan, Bangkok, 10330
Thailand, 4 Department of Animal Production and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang,
Chalongkrung Rd., Ladkrabang , Bangkok 10520 Thailand
Volume 50 Issue 3 (2016)
were good in fertility profiles and were used as semen donors
to produce commercial frozen semen.
Sperm quality assessment: The volume of fresh semen was
measured in milliliter (ml) using a collective tube at the time
of collection. The concentration of each ejaculation was
determined by a photometer (Sperma CueTM, MinitÜb
GmbH, Tiefenbach, Germany). The individual progressive
motility of sperms were monitored and assessed subjectively
by bright field microscope. The viability and morphology
of sperms were evaluated independently under a light
microscope (1000x) after eosin-nigrosin staining (Dott and
Foster, 1972). A total of 200 sperms per ejaculate were
evaluated in each attributes. Sperm with an unstained head
were regarded as a live sperm. Sperm with a crescent shaped
apical ridge were considered as morphologically abnormal.
7-Layer Percoll®density gradient centrifugation: Sperm
preparation by 7-layer Percoll® gradient was developed by
Kobayashi et al. (2004). A modified discontinuous 7 layer
technique was used for the Percoll® separation method in
order to obtain an isotonic medium upon dilution with 100%
Percoll®. The Percoll® stock solution was diluted further to
obtain 80, 75, 70, 65, 60 50 and 40% (v/v) Percoll® solution.
Percoll® solution (1.0 ml aliquots) in 15 ml Falcon ® tube
were layered from higher to lower concentration, starting
with 80% at the bottom. The sperm specimen was laid onto
the top of the upper Percoll® layer (40%). The tube was
centrifuged for 15 min at 300xg. The 70 and 65% fraction
containing sperms pellet was collected (after removing the
upper layers) by disposable pasteur pipette and washed with
Tris buffer with egg yolk. The recovered spermatozoa were
resuspended in 0.5 ml of medium and processed to frozen
for further use.
Identification of Y-bearing sperm by quinacrine
staining: 0.1% solution of quinacrine mustard with cold
distilled water was freshly prepared before testing. 7 l of
quinacrine stain was mixed on a glass slide with 7 l of
sperm specimen. All slides were monitored immediately
using fluorescent microscopy. Head of sperms which
showed fluorescent round body were counted as Y-bearing
sperm. A raw semen sample was counted for 50 ± 2% Ybearing sperm as a stain control.
Sperm freezing and thawing: Sorted sperms were frozen
with Tris egg yolk extender using the freezing protocol
modified from Anzar et al. (2011). Briefly, samples were
extended at room temperature in Tris-egg yolk extender
(3.0 g Tris, 1.7 g citric acid, 1.3 g fructose, 0.1 g penicillinstreptomycin, 8% (v/v) glycerol and 20% (v/v) egg yolk)
to obtain a final concentration of 1.2 x 10 8 sperms/ml. The
diluted samples were gradually chilled to 4 oC and further
kept at this temperature for 4 h. Then samples were filled
into mini-straws (0.25 ml) and froze in astyro-foam box
contained dry ice and transferred into liquid nitrogen.
Samples were kept under liquid nitrogen at least 1 day
311
before thawing. In the throwing process, straws were dipped
into 37oC water for 30s. The thawed sperms were used
immediately for AI.
Artificial insemination: Cows found in standing heat were
submitted to 6-hourly ovary scans until ovulation was
confirmed. Kept water bath at 34-35 °C ready. Then sperm
on a layer of 65-70% Percoll® have been thawed. All sperms
in this layer were used for artificial insemination. Information
for each was recorded at the time of A.I. Similar age cows
were let loose to pasture together and an Angus bull
introduced 3 weeks after AI to cover any heifers that returned
to oestrus. Heifers had EstrotectTM heat detectors applied on
Day 17 post-oestrus and were checked for activation every
alternate day until Day 26. Cows were scanned using
transrectal ultrasonography at 52 days post insemination to
confirm pregnancy. The information of pregnancies have
been monitored until calving. All information of new calves
born under AI using fresh prepared sexing sperm and freezethaw sperm were compared with calves born under nonsexing method.
Embryo transfer: Bovine ovaries were collected from
slaughter house in Bangkok area and transferred to 0.9%
NaCl until used. At the same day of ovary collection, oocytes
were collected, selected and taken under stereo microscope.
Selected oocytes were cultured under TCM199 medium with
10% FBS. Mature oocytes were collected and co-incubate
with 5 million sperms which have been selected by Percoll®
centrifugation. Co-incubation have been done under
TCM199 with 5% calf serum under 38°C for 12 hours. The
In vitro fertilized oocytes were then washed and continued
culture for 2 days. 8 cell stage oocytes were selected and
transferred to mSOF medium under 38.5°C and cultured for
5 days until blastocyst. Embryos were transferred to surrogate
mothers at Pakthongchai Dairy Research Farm and operated
under the same protocol as AI cows. All information of calves
born under ET method have been collected and compared
with calves born under AI and non-sexing method.
Statistical analysis: Statistical Package for the Social
Sciences (SPSS Statistics 17.0, Chicaco, IL) was used for
statistical analysis. The sperm parameters were presented as
Mean ± Standard Error of the Mean (SEM). Differences in
the percentages of sperm motility, viability, and the cleavage
rates of embryos among each experimented groups have been
analyzed with one-way ANOVA. When ANOVA showed a
significant effect, values were compared using the Least
Significant Difference (LSD) test. The level of significance
was defined as P<0.05.
RESULTS AND DISCUSSION
This experiment was conducted with the
collaboration of Pakthongchai Dairy Research Farm, Nakhon
Ratchasima, Thailand. Semen has been collected from
Holstein bulls and centrifuged on a seven layer of alignment
as 40%, 50%, 60%, 65%, 70%, 75% and 80% of Percoll®.
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INDIAN JOURNAL OF ANIMAL RESEARCH
All quality and quantity of sperms have been monitored
before and after the process. Semen qualities before and after
Percoll® separation are shown in Table 1. All sperms in 40%
Percoll® solution layer have been discarded because no
significant motile sperm was monitored. We have found only
few sperms can swim into 4 layers of 65% to 80% Percoll®
concentration. Therefore, sperms in 65% and 70% was
combined similar as sperms in 75% and 80%.
There were significant differences of sperms
motility and phenotype among each layer after separation.
As shown in Table 2, the means of X-bearing spermatozoa
percentage floated between 75% and 80% layers were
76.21%. However, these sperm showed low motility as only
22.57±0.59% (Table 1). Therefore, only sperms on 65-70%
layer were selected for further progression (60.75%
X-bearing spermatozoa and 95.86±0.46% motility).
At the farm, sperms have been prepared within 30
minutes before AI. The average percentages of quantity and
qualities of sperm in 65-70% Percoll® layer from these
frozen-thawed sperm were shown in Table 3.
®
The efficiency of the Percoll gradient media in
separation of X-bearing spermatozoa was evaluated directly
through sex ratio of the offspring from ET or AI as shown in
Table 4. In embryo transfer, the separated sperms were used
for in vitro fertilization (IVF). A total of 178 fertilized eggs
were produced from 201 mature oocytes (88.55%). A total
of 69 blastocysts were developed and transferred to recipient
cows. A total of 28 cows were pregnant (40.58%). After 9
months of pregnancy, the calves were born with 19 females
(67.86%) and 9 males (32.15%). On the other hand, the AI
was done in 150 cows, 60 cows were pregnant but only 42
calves were born with 30 females (71.43%) and 12 males
(28.57%). The results have been compared with non-sexing
sperm as shown in Table 4.
The objective in this experiment was to determine
the sperm enrichment with X-bearing spermatozoa, after
centrifugation in a Percoll® discontinuous density gradient,
through the ratio of a number of offspring. This protocol
have been developed to reduce the cost of sperm preparation
and simplify for farmer to use in a small commercial dairy
farm.
The quality of sperm after centrifugation in each
layer were significantly different (P <0.01). The number of
sperm and group motivation were lower when increased
Table 1: Sperm characteristics before and after passage through Percoll® gradient.
Sperm parameter
Fresh
spermatozoa
6
a
Concentration (×10 ml)
Progressive motility (%)
Viability (%)
Normal morphology (%)
852.93±30.2
92.50±0.51b
92.71±0.69ab
46.07±0.64a
After passage through a Percoll® gradient (%)
60
65-70
50
c
138.86±6.51
84.00±0.55d
91.36±0.95c
35.57±0.55c
c
144.14±5.04
90.29±0.62c
93.43±0.44a
33.64±0.43d
414.93±30.32
95.86±0.46a
93.57±0.57a
39.07±0.44b
b
75-80
35.29±1.05d
22.57±0.59c
78.50±0.58c
33.57±0.40d
Data are expressed as mean percentage ± SEM. Different letters indicate significant difference within a row (p< 0.01).
Table 2: The percentage of X-bearing spermatozoa in different
layers of Percoll gradient.
Percoll gradient
layer (%)
No of X-bearing
spermatozoa (%)a
0
50
60
60-75
75-80
49.75
39.36
41.43
60.75
76.21
Table 3: In vitro quality of fresh and frozen-thawed bull
sperm at ozoa aft er passage t h rou gh a P ercoll gradien t
65-70%
Sperm parameter
Passage through a Percoll gradient (%)
Fresh
spermatozoa
Concentration (×106 mL)
Motility (%)
Viability (%)
Frozen-thawed
spermatozoa
414.92
94.29
94.57
8
65.86
69.14
a
The percentage of X-bearing spermatozoa enrichment, as determined by
quinacrine staining.
Data are expressed as mean percentage ± SEM.
Table 4: Effects of PercollTMtreatment on sex (%) of offspring after AI and ET.
Treatment
ET
AI
AI with nonsexing sperm
No. of transfer
69
150
128
No. (%) of pregnant cows a
28 (40.58%)
60 (40.00%)
59 (46.09%)
Sex (%) of offspring
male
9 (32.15%)
12 (28.57%)
29 (49.15%)
female
19 (67.86%)
30 (71.43%)
30 (50.85%)
Data from three separated experiments have been presented as number (percentage) of pregnant cows. aindicates non-significant difference of percentage
within column.
Volume 50 Issue 3 (2016)
Percoll® concentration. However, the abnormality phenotype
of sperms were lower in higher concentration. Percentage
of sperm moving forward with increase in pH was
significantly higher (P <0.01). There was no significant
difference between percentage of live sperm after
centrifugation compared with fresh sperm before
centrifugation (P >0.05). Thus, the layer in Percoll ®
centrifugation can increase the quality of semen.
After gradient centrifugation, the ratio of X-sperm
in the samples selected by Percoll® did not significantly alter
in comparison to the non-centrifuged samples (Table 2). This
result was accordance with some previous findings
(Kobayashi et al., 2004) that using multilayer Percoll ®
gradients which could successfully enrich X-bearing bovine
sperm.
After insemination, the pregnancy ratio in this
experiment was 40% which was not different from the record
of Pakthongchai Dairy Research Farm which indicated the
313
conception rate of AI as 30-40% (Table 4). Therefore, the
used Percoll ® gradient did not influence the pregnancy
percentage whereas selected sperms had increased the
number of female offspring up to 71.4% where Pakthongchai
Dairy Research Farm recorded the ratio of male-female as
50-50% without sperm selection. In the In Vitro fertilization
experiment, sperm from our 7 layer Percoll ® gradient
concentration can fertilize and produce blastocyst embryo
successfully. Thus, our developed 7 layer Percoll® gradient
concentration can be used as a simple protocol for farmer to
reduce the cost and increase the number of female calves in
their dairy farm.
ACKNOWLEDGEMENTS
The authors are grateful to Graduate Study and
Research in Agricultural Biotechnology Sub project for
financial support. We also wish to thank Paktungchai dairy
farm for supplying bovine semen and dairy cows for this
study.
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