MINISTRY OF EDUCATION
AND TRAINING
MINISTRY OF AGRICULTURE
AND RURAL DEVELOPMENT
NATIONAL INSTITUTE OF ANIMAL SCIENCES
HA MINH TUAN
STUDY ON PRODUCTION OF FROZEN SEMEN STRAW
OF VIETNAMESE NATIVE BUFFALO BULLS
Major: Animal production
Code No.: 62 62 01 05
SUMMARY OF PhD THESIS
HANOI - 2014
The thesis was completed at: National Institute of Animal
Sciences
Supervisors:
1. Associate Professor, Doctor Mai Van Sanh
2. Doctor Le Van Thong
Opponent 1: ……………………………………..
Opponent 2 ………………………………………
Opponent 3: ………………………………………
The thesis is sumitted before the Thesis Examination
Committee (Institute Level) meeting at the National Institute of
Animal Sciences at
[Time] [Date]
[Month]
[Year]
Thesis can be found at:
1. National Library
2. Library of National Institute of Animal Sciences
3. Library of Vietnam Ruminant Breeding Center
1
INTRODUCTION
1.RATIONALE
Many countries have paid much attention on buffalo research
such as: India, Pakistan, China, Brazin...and gained the succeeses in
development and improvement of buffalo production through the
artificial insemination programe and frozen straw semen production.
(Anzar et al., 2003; Liang et al., 2004; Vale, 2010; Bhakat et al.,
2011).
Until now, there have not been any authors, who
systematically studied straw semen production under Vietnamese
conditions. Thus, it is logical and necessary to conduct a “Study on
production of frozen straw semen of Vietnamese native buffalo
bulls”.
2. OBJECTIVES OF THE STUDY
To evaluate some characteristics of semen quantity, quality
and find out a suitable extender and methods of semen freezing. At
the same time, the frozen semen production potential of buffalo bulls
is estimated.
3. SCIENTIFIC AND PRACTICAL VALUES OF STUDY
3.1. Scientific values:
The result of this thesis will contribute and enrich a data base
on buffalo bull training, semen collection by artificial vagina, age at
first semen collection, characteristics of semen quantity and quality,
potential of frozen semen production, suitable extender for semen
freezing, a better way of semen freezing and influence of season on
semen quality of swamp buffalo bulls in the North.
2
This thesis can also be a scientific document for reference at
research institutes, universities, colleages, as well as breeding buffalo
bull farms.
3.2. Practical values
- The production of frozen straw semen with a high quality
will support the artificial insemination in Vietnam, and contribute to
improvement of quality and quantity in swamp buffalo development.
- Understanding possible influence of season on semen
production is very helpful for breeding buffalo bull farms.
4. NEW CONTRIBUTIONS OF THE THESIS
The thesis is the first scientific work to assess some
characteristics of semen quantity and quality, potential of frozen
semen production of Vietnamese native buffalo bulls. This thesis is
also the first scientific work to assess possible influences of season
on semen production of buffalo bulls in the Northern environmental
condition in Vietnam.
The thesis also determines a suitable extender and way of
semen freezing to produce frozen semen in Vietnam.
Thesis also the first scientific work to confirm that Vietnam
can produce high-qualified straw frozen semen for swamp buffaloes
breeding programs.
Charpter 1. LITERATURE REVIEW
1.1. INTRODUCTION OF VIETNAMESE NATIVE BUFFALO
Vietnamese native buffalo is swamp buffalo (Berthouly et al.,
2010), kept mainly in two midland areas: Northest mountain
(occupied by 56.77%) and North Central Coast (occupied by
30.54%) (Department of livestock production, 2010).
3
1.2. SEMEN COLLECTION AND QUANTITY, QUALITY OF
BUFFALO SEMEN.
About ruminant, the semen collection in a artificial vagina is
the most popular mrthod. (Dorji, 2009).
Semen volume of Murrah buffalo in India from 2.31 to 7.36
years old has been reported to be 2.58ml (Bhakat et al., 2011).
Nordin et al. (1990) reported that volume of semen for the swamp
buffalo from 29 to 65 months old ranges from 1.5-3.7ml.
Aguiar et al. (1994) observed that there has been 76.8%
motile spermatozoa in the semen of buffalo bulls in the Brazilian
states. Kumar et al. (1993a) found that in semen of Murrah buffalo
bulls in India, motile spermatozoa varied from 60.8% to 69%.
Vale (1994a) reported that sperm concentration in buffalo
semen varied from 0.6 to 1.2 x 109 cells/ml. Bhakat et al. (2011)
informed that Murrah buffalo bull had the average sperm
concentration of semen 0.99 x 109 cells/ml.
1.3. FACTORS AFFECTING QUANTITY AND QUALITY OF
BUFFALO SEMEN
Sperm quantity and quality are affected by many factors such
as: breed, individual, age, season, food, management of semen
collection (Nazir, 1988; McCool et Entwistle, 1989; Javed et al.,
2000, Shukla et Misra, 2005).
1.4. FROZEN STORAGE OF SEMEN
There have been many cryoprotectants, which have been used
as extenders of semen such as: tris, citric acid, natri citrate, glucose,
fructose, lactose, raffinose, penicillin G and streptomycin, glycerin, egg
yolk. (Singh et al., 1995; Siddique et al., 2006; Vale, 2010; Bansal et al.,
2011).
4
Freezing semen in liquid nitrogen could pratically be done by
using a simple isothern box. Straws are suspended in horizontal
position 1-4cm above liquid nitrogen for 10-20 min, then they are
plunged and stored into liquid nitrogen at -196oC (Ansari et al., 2011).
Freezing semen in liquid nitrogen could also be done by using
professional prorgamed equipment systems (Anwar et al., 2008).
1.5. ARTIFICIAL INSEMINATION OF BUFFALO BY FROZEN
SEMEN.
According to Haranath et al. (1990), using the extender
combined of egg yolk, tris and glycerol for freezing buffalo semen in
2 types of straws (0.25 ml and 0.5 ml), resulted in conception rates of
52.7 and 50.4%. Using extender of Tris-citric acid (TCA) for
freezing buffalo semen, resulted in a conception rate of 56.75 in NiliRavi (Andrabi et al., 2006).
1.6. LOCAL AND FOREIGN RESEARCH
India, Pakistans, Brazil, China and Thailand have produced
frozen semen for local buffalo development for a long time
(Bhattacharya, 1955; Vale et al., 1984; Liang Xian wei et al., 2004).
The artificial insemination system in Philipin could provide 55,000
frozen semen straws per year, and could coverd approximately 5%
local female buffalo. (Cruz, 2006).
Luu Ky (1979), Vu Ngoc Ty and Luu Ky (1979) have made
a success in semen freezing on the surface of liquid nitrogen. Le Viet
Anh et al. (1984), Nguyen Huu Tra et al. (2001) have produced
frozen semen tablets of Murrah buffalo bulls using extenders of bulls
(Nagase) and Triladyl imported from Germany. Post thawing motility
of the above frozen semen tablets was 35% and conception rate of
buffalo females was approximately 50% (double service). When
5
using 3 extenders: Citrat-L, Citrat-G and TCA for freezing bufflo
semen, Trinh Thi Kim Thoa (2006) found out that post thawing
motility of these frozen semen straws was 22.5%, 37.5% and 42.5%,
respectively.
Charpter 2. MATERIALS AND METHODS
2.1. ANIMALS
- Six Vietnamese native buffalo bulls and 150 local buffalo
cows were used in this study.
2.2. LOCATION AND DURATION
- Study was undertaken in i) Moncada Station for Research
and Production of frozen semen (Address: Tan Linh, Ba Vi, Hanoi);
ii) Bim Son town and Ha Trung district, Thanh Hoa province and iii)
Thanh Chuong district and Tan Ky district, Nghe An province.
- Study was conducted from January 2011 to December 2013.
2.5. CONTENTS OF STUDY
- Buffalo bull training and semen collection.
- Influence of individual and season on characteristics of
semen quantity and quality.
- Choosing the suitable dilution extender and freezing methods.
- Assessing potential of frozen semen straw production.
- Testing quality of straw frozen semen.
2.6. METHODS
2.6.1. Buffalo bull training and semen collection.
- Semen was collected by artificial vagina.
- Age to be trained for semen collection and age for semen
collection in Vietnam buffalo was monitored, recorded through
check-list of each bull.
6
- Body weight of buffalo was weighted using an electric scale.
- Training duration of bull was measured by observation of
total days from the first day of training to the day in which buffalo
bull has a good skill to jump and semen can be fully collected using
an artificial vagina.
2.6.2. Influence of individual and season on characteristics of
sperm quantity and quality.
Semen volume per ejaculate was measured directly from
graduated collecting tube and recorded in ml.
Sperm motility was measured using a micropipet to suck out
0.01ml semen and 0.09 ml A liquid (without glycerol); then droped it
on dry glass slide and maintained at 38oC, covered slide with a lamen
and placed it into Microscope Depot with magnification of 100 fold
linked to display interface. Sperm motility was assessed by 3
different experts (Jainudeen et al., 1982; Beheshti et al., 2011).
- Sperm concentration: Using a micropipet to suck out
0.02ml semen dilution, droped it into 4ml Natri Clorid 0,9%, gently
shaked and placed it into Photometer SDM5 (Minitub, Germany).
Concentration of semen (x 109 cells/ml) was read on the screen of the
eqipment.
- Abnormally shaped sperm: Using Fucsin 5% to dye
spermatozoa in 5 to 7 min, put dyed sperm on a dry grass slide and
counted number of normal and abnormaly shaped sperm/500 sperms
with a Microscope Depot of a magnification of 400 fold.
The
abnormally shaped sperm percentage was:
Abnormally shaped sperm
percentage (%) =
Numbers of Abnormally
shaped sperms
500
x 100
7
- Live sperm (%) was measured using Blom method (1950).
First, put 01 drop of and 02 drops of Eosin 5% semen into a dry deep
slice, slightly mixed, then put 04 drops Nigrosin 10%, slightly mixed
again, maintained a mixture at 370C for 30 seconds. One drop of
dyed semen mixture was then taken and put on a glass slide, and
counted number of normal and abnormaly shaped sperm/500 sperms
with a Microscope Depot of a magnification of 400 fold. (dead sperm
turned red with Ecosin). The live sperm percentage was:
Live sperm
percentage (%) =
Numbers of live sperms
500
x 100
2.6.3. Choosing dilution extender and freezing methods
- The sample of sperms for this experiment was qualified
(volume of semens was not less than 1ml, sperm motility was not less
than 70%, sperm concentration was not less than 0,6 x 109 cells/ml,
abnormally shaped sperm percentage was not more than 20%, live
sperms percentage was not less than 70% (Herdis et al., 1999;
Koonjaenak, 2006; Vale, 2010; Ansari et al., 2011, Swelum et al.,
2011, El-Kon, 2011). Qualified samples of sperm of each collection
and of each bull were mixed together (Rasul et al., 2000, Swelum et
al., 2011, El-Sheshtawy et al., 2013), then this mixture was divided
into 6 equal parts for the experiment, in which 3 extenders and 2
methods of freezing.
- Three extenders: i) Extender 1 (Ext 1) of Pakistan (1.21g
Tris, 0.67g axit Citric, 1.04g Natri citrate, 0.25g Fructose, 0.25g
Glucose, 1g Lactose, 100.000UI Penicillin G, 100mg Streptomycin,
7% glycerin, 20% egg yolk and distilled water) (Siddique et al.,
2006); ii) Extender 2 (Ext 2) of India (3.028g Tris, 1.675g axit Citric,
1.25g Fructose, 100.000UI Penicillin G, 100mg Streptomycin, 7%
8
glycerin, 15% egg yolk and distilled water) (Singh et al., 1995); iii)
Extender 3 (Ext 3) of Japan (1.363g Tris, 0.762g axit Citric, 0.375g
Fructose, 1.5g Lactose, 2.7g Raffinose, 100.000UI Penicillin G,
100mg Streptomycin, 6.5% glycerin, 20% egg yolk and distilled
water) (Dao Duc Tha et al., 2007, 2010) were used.
- Two methods of freezing: i) Fast freezing (PP1) and slow
freezing were used.
For fast freezing (PP1): Semen straw was placed into a tray in a
horizontal direction with a distance of 4cm from nitorgen surface (1200C) in 10 minutes, then straws were put into liquid nitrogen (1960C) for preservation (Verma et al., 1975; El-Sheshtawy et al.,
2008, Akhter et al., 2011; Ansari et al., 2011).
For slow freezing (PP2): Freezing temperature was programed to be
continuously and graduately reduced. First, temperature was reduced
from 40C to -60C with a speed of 30C/min, then reduced from -60C to
-700C in 8 min, from -700C to -1650C with a speed of 240C/min.
Finally, frozen straws were put into liquid nitrogen tanks at -1960C.
(Phung The Hai et al., 2011).
- After 24h freezing, 03 frozen semen straws of each freezing
method and extender were randomly taken, thawed at 370C in 30
seconds (Siddique et al., 2006, Andrabi et al., 2008; Ansari et al.,
2011). Finally, characteristics of post thawing sperm were checked
for sperm motility, concentration and live sperm perentage.
2.6.4. Method of accessment of capacity of frozen semen straw
production.
Potential of frozen semen straw production was measured
using number of good quality semen straws produced by each bull.
2.6.5. Testing frozen semen straw quality.
9
Post-thawing sperm motility (%) was determined after 24
hours storage on 01 frozen semen straws randomly taken for each
semen straw production bath and each standardized collection.
Quality of post-thawing semen was examined using Microscope
Depot as previously mentioned.
Rectal palpation after 90 days of insemination was used for
conception rate of buffalo cows inseminated with frozen semen
straws produced fron this study.
Number of pregnant buffalo
Conception rate (%)
=
cows
x 100
Total inseminated buffalo cows
2.7. STATISTICAL ANALYSIS
- Before statistical analysis, data expressed in a form of
percentage such as motility of semen, abnormally shaped sperm
percentage, live sperm percentage were changed to another form
using the following equation:
Y = Degrees {asin[sqrt(x/100)]}.
After statistical analysis, these data were changed to the
orignal form: Y 1 = 100 x {Sin[radians(x)]} 2 (Nguyen Van Duc and
Le Thanh Hai, 2002).
Data on quantity, quality of buffalo semen (volume of
semens, sperm mitility, pH of semens, abnormally shaped sperm, live
sperm percentages), semen straw production, standardized semen
straws, quality of frozen semen straw were analyzed by ANOVA,
one-way. Differences between two mean were checked using Tukey
in Minitab 14 .
Percentage of high-qualified semen straw production of each
individual and of each season, conception rate were compared using
Chi-square Test.
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Charpter 3. RESULTS AND DISCUSSION
3.1. BUFFALO BULL TRAINING AND SEMEN COLLECTION
Results of buffalo bull training and collection of buffalo
semen by a artificial vagina in three times per week, showed that
total time for training for buffalo semen collection was 30 days, in
which 6 days for the bull number 301 and 59 days for the bull
number 307. The reason was that buffalo bulls were different in ages
and body weight.
Buffalo bull body weight at starting time of collection was
518.50kg. This body weight was higher than that reported by Mai
Van Sanh (2005) and Nguyen Cong Dinh (2012).
In conclusion, Vietnam buffalo bulls in this research had
heavy body weight. They also had a high mating reflect for semen
collection by an artificial vagina.
Table 3.1. Duration of training and semen collection of buffalo bulls
Starting time of
Bull
no.
training for
Duration of training to
Starting time for
semen
collect semen
semen collection
collection
Age
(month)
Weigh Adaptation
(kg)
(days)
Training
to jump
(days)
Sum
Age
Weight
(days) (months) (kg)
301
31.00 562.00
3.00
3.00
6.00
31.50
571.00
302
31.00 514.00
4.00
5.00
9.00
32.00
537.00
304
26.00 469.00
10.00
18.00
28.00
28.50
515.00
305
26.00 423.00
11.00
22.00
33.00
29.00
508.00
306
25.00 413.00
15.00
30.00
45.00
28.50
486.00
11
307
25.00 407.00
20.00
39.00
59.00
29.50
494.00
Mean 27.33 464.67
10.50
19.50
30.00
29.83
518.50
6.47
14.01
20.47
1.54
31.20
SD
2.88
62.70
3.2. INFLUENCE OF INDIVIDUAL AND SEASON ON SEMEN
QUANTITY AND QUALITY .
3.2.1. Influence of individual on semen quantity and quality of
Vietnamese native buffalo bulls.
3.2.1.1. Volume
According table 3.2, average volume of semen of buffalo
bulls was 3.89 ml, varied from 3.16ml of bull number 306 to 4.50ml
of bull number 302 (P<0.05). Results of this study were similar to
results of Shukla and Misra (2005), Khawaskar et al. (2012) and
Mahmoud et al. (2013). These authors also found differences in
semen volume among individual buffalo bulls.
Table 3.2. Semen volume of Vietnamese native buffalo bulls
Bull no.
Volume (ml)
Number of
collection (times)
Mean
SD
301
120
4.07b
0.30
302
120
4.50a
0.32
304
120
4.41a
0.27
305
120
3.81c
0.29
306
120
3.16e
0.27
307
120
3.42d
0.32
Average
720
3.89
0.57
12
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
Semen volume of Vietnamese native buffalo bulls was
similar to that of bulls in Malaysia, Thailand, and of Nili-Ravi
buffalos. According to Nordin et al. (1990), average semen volume in
Malaysian swamp buffalo bulls ageing from 54-65 months old was
3.5ml. Volume of semen of swamp buffalo bulls used in artificial
insemination in Thailand from the period of 2004 to 2005, was 3.6ml
(Koonjaenak et al., 2006). Sajjad et al. (2007) studied bulls of NiliRavi breed and found that volume of semen was 3.59ml.
3.2.1.2. Sperm motility
Table 3.3. Sperm motility of Vietnamese native buffalo bulls.
Sperm motility (%)
Number of
collection (times)
Mean
SD
301
120
75.20bc
4.22
302
120
73.52c
4.05
304
120
78.26b
4.38
305
120
81.50a
4.95
306
120
71.24cd
3.87
307
120
70.08d
3.62
Average
720
75.08
4.51
Bull no.
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
Average sperm motility of Vietnamese native buffalo bulls
was 75.08%, varied from 70.08% in bull number 307 to 81.50% in
13
bull number 305 (P<0.05). Sperm motility of Vietnamese native
buffalo bulls in this study was similar to that of buffalo bulls in
Malaysia, Thailand, Murrah and Azarbaijani buffalos. Swamp
buffalo bulls of Malaysia aging more than 65 months old had the
sperm motility of 75.4% in age of 65 months (Nordin et al., 1990).
Nair et al. (2012) found that sperm mobility was 77.5%. According to
Shukla et Misra (2005) sperm mobility of Murrah buffalo bulls, was
77.92%. Alavi-Shoushtari and Babazadeh-Habashi (2006) found that
sperm motility was 75.85%. Koonjaenak et al. (2006) found out that
sperm motility of swamp buffalo bulls used for artificial insemination
in Thailand from the period of 2004 to 2005 was 73.4%.
3.2.1.3. Sperm concentration
Table 3.4. Sperm concentration of Vietnamese native buffalo
bulls
Number of
Bull no.
collection (times)
Sperm concentration (x 109 cells/ml)
Mean
SD
301
120
1.04d
0.09
302
120
1.20b
0.09
304
120
1.11c
0.10
305
120
1.14c
0.10
306
120
1.44a
0.11
307
120
0.93e
0.09
Average
720
1.14
0.18
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
14
Average of sperm concentration of Vietnamese native buffalo
bulls (1.14 x 109 cells/ml) was similar to that of buffalo bulls in many
other countries. Sperm concentration of swamp buffalo bulls in Malaysia
was 1.06 x 109 cells/ml (Jainudeen et al., 1982) and sperm concentration
of swamp buffalo bulls in Indonesia was 1.18 x 109 cells/ml (Herdis et
al., 1999). Swamp buffalo bulls used for artificial insemination in
Thailand from the period of 2004 to 2005 had a sperm concentration of
1,1 x 109 cells/ml (Koonjaenak et al., 2006). Sperm concentration of
Vietnamese native buffalo bulls also varied from the highest one in bull
number 306 (1.44 billion/ml) to the lowest one in bull number 307 (0.93
billion/ml) (P < 0.05). Variation in sperm concentration of Vietnamese
native buffalo bulls was similar to that reported of Shukla and Misra
(2005). They found that sperm concentration of Murrah buffalo varied
from 0.92 billion/ml to 1.24 billion/ml (P<0.05).
3.2.2. Influence of season on sperm quantity and quality
Semen volume of buffalo bulls was the highest in autumn
and winter (4.07ml and 4.13ml). Semen volume of buffalo bulls in
spring was lower (3.8ml) and semen volume of buffalo bulls in
summer was the lowest 3.56ml (P<0.05). It seemed that difference in
temperature among seasons affected semen production of buffalo
bulls.
In spring, low temperature and high humidity affected
reproduction of bulls. This could also affect reproduction of bulls in
summer. In autumn, stable temperature and humidity made better
semen quality.
Table 3.9. Volume of Vietnamese native buffalo bulls in each
season
Season
Number of
Volume (ml)
15
collection (times)
Mean
SD
Spring
180
3.80b
0.54
Summer
180
3.56c
0.54
Autumn
180
4.07a
0.50
Winter
180
4.13a
0.53
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
Sperm motility of buffalo bulls was the highest in winter
(77.88 %). Sperm motility of buffalo bulls was 75.94 and 75.03% in
spring and autumn, respectively. Sperm motility of buffalo bulls was
the lowest in summer (71.32%).
Many other authors reported similar results. Mandal et al.
(2000) reported sperm motility of Murrah bull semen in winter was
the highest. According to Al-Sahaf and Ibrahim (2012), semen
quality and quantity of buffalo bulls increased in months with low or
medium temperature and reduced in months with high temperature.
Igna et al. (2010) reported that temperature affects not only day of
collection of sperm but also process of spermatozoa production in
testicular.
Table 3.10. Sperm motility of Vietnamese native buffalo bulls in
each season
Season
Number of
Sperm motility (%)
collection (times)
Mean
SD
Spring
180
75.03b
4.10
Summer
180
71.32c
4.41
16
Autumn
180
75.94ab
3.96
Winter
180
77.88a
4.09
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
Sperm concentration was the highest in winter (1.24 x 109
cells/ml), followed that in autumn (1.19 x 109 cells/ml), and in spring
(1.11 x 109 cells/ml). Sperm concentration was the lowest in summer
(1.03 x 109 cells/ml) (P<0.05).
Results of thie study were in aggrement with several works.
Sperm concentration of Nili-Ravi buffalo semen in Pakistan was high in
autumn and spring (Javed et al., 2000). Sperm concentration of Murrah
buffalo semen was the lowest in summer (Gokhale et al., 2003). Sperm
concentration of Taiwan buffalo bulls was high in autumn and spring
(Wei et Jea, 2006). Sperm concentration of Iraqui buffalo bulls increased
in months with low and medium temperaature and reduced in months
with high temperature (Ibrrhem et al., 2014).
Table 3.11. Sperm concentration in Vietnamese native buffalo
bull each season
Season
Number of
Sperm concentration
collection
(x 109 cells /ml)
(times)
Mean
SD
Spring
180
1.11c
0.16
Summer
180
1.03d
0.15
Autumn
180
1.19b
0.17
Winter
180
1.24a
0.18
17
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
In conclusion, semen quantity and quality of Vietnamese native
buffalo bulls was similar to these of swamp buffalo in Thailand,
Malaysia, Indonesia. Individual and season affected semen quantity and
quality of Vietnamese native buffalo bulls.
3.3. CHOOSING EXTENDER AND FREEZING METHOD
As can be seen from tables 3.17 and 3.18, quality of frozen
semen in extender Ext 3 was better than that of extender Ext1 and
Ext (P<0.05) in the fast freezing method (PP1). In the slow freezing
method (PP2), quality of frozen semen in extenders Ext 1 and Ext 2
was similar (P>0.05) and quality of frozen semen in both Ext 1 and
Ext 2 was lower than that in extender Ext (P<0.05).
Post thawing semen quality with extender Ext 3 was higher
than that with extenders Ext 1 and Ext 2 in both fast freezing (PP1)
and slow freezing methods (PP2) (P<0.05). This result was similar to
works done by other authors who studied extender with raffinose.
Stoianov and Kostadinov (1978) used extender with the high amount
of raffinose, without glycerol, and found out that results was similar
compared with extender with glycerol. Dhami and Sahni (1993)
reported that post thawing semen quality was still good when
extender included Tris, fructose, raffinose, egg yolk and glycerol was
used.
For all 3 extenders Ext 1, Est 2 and Est 3, post thawing
semen quality in the slow freezing method (PP2) was higher than that
in fast freezing method (PP1) (P<0.05). Bhosrekar et al. (1994) and
Narayan et al. (2000) reported that quality of ruminant sperm in a
slow freezing method using a programmed refrigerator was higher
18
than that in fast freezing method with liquid nitrogen. a quick
reduction in temperature caused a cold shock to sperms (Andrabi,
2009) and cell of sperms (Watson, 2000), therefore quality of frozen
semen reduced.
Table 3.17. Post thawing quality of semen of Vietnamese native
buffalo bulls in 3 extenders using fast freezing method (PP1)
Post thawing quality of semen
Extender
Motility (%)
Abnormally shaped
Live
sperm (%)
sperm (%)
Mean
SD
Mean
SD
Mean
SD
Est1
40.10a
0.62
22.57b
0.33
57.86ab
0.43
Est 2
39.07
a
0.69
23.88
b
0.32
55.37
a
0.48
Est 3
45.89b
0.54
18.64a
0.19
61.37b
0.70
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
Table 3.18. Post thawing quality of semen of Vietnamese native
buffalo bulls in 3 extenders using slow freezing method (PP2)
Post thawing quality of semen g
Extender
Motility (%)
Mean
46.01
a
Est 2
44.90
a
Est 3
51.97b
Est1
SD
Abnormally shaped
Live
sperm (%)
sperm (%)
Mean
SD
Mean
18.33
b
0.51
20.16
b
0.23
61.09
0.71
15.53a
0.21
68.79b
0.59
0.34
SD
63.19
a
0.38
a
0.67
0.55
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
19
In conclusion, quality of frozen semen straw was the best
when extender Ext 3 and slow freezing method were applied (PP2).
3.4. POTENTIAL OF PRODUCTION OF FROZEN SEMEN
STRAW
3.4.1. Potential of production of frozen semen straw of
Vietnamese native buffalo bulls.
It was revealed that average number of semen straw
produced from Vietnamese native buffalo bulls was 152.56
straw/standardized
collection.
Number
of
semen
straws
produced/standardized collection was the highest in bull number 302
(183.96 straw/standardized collection and was the lowest in bull
number 307 (110.58 staw/standardized collection). (P<0.05).
Table 3.23. Semen straw production of Vietnamese native buffalo
bulls produced per standard collection
Number of
Number of straw
standardized
Standardized production/standardized Standard straws/standardized
Percentage
collection/bull
Bull no. collection
collection collection/bull
(%)
(straw)
(times)
(times)
(straw)
Mean
SD
Mean
SD
301
87
144.17c
20.52
81
143.58c
20.41
93.10
302
81
183.96a
20.46
74
185.16a
20.86
91.36
304
101
166.80b
21.40
91
166.81b
20.38
90.10
305
108
147.40c
22.31
101
147.94c
22.00
93.52
306
75
152.99c
23.81
66
153.42c
25.00
88.00
20
307
65
110.58d
19.81
56
109.80d
20.82
86.15
Average
517
152.56
29.85
469
152.94
30.08
90.72
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
3.5. FROZEN SEMEN STRAW QUALITY OF VIETNAMESE
NATIVE BUFFALO BULLS.
3.5.1. Post thawing sperm motility of Vietnamese native buffalo
bulls
Post thawing sperm motility of Vietnamese native buffalo bulls was
49.17% on average. However, post thawing sperm motility of
Vietnamese native buffalo bulls varied from the highest value in bull
number 305 (52.03%) to the lowest values in bull number 306 and
307 (47.24% and 49.17%) (P<0.05). Post thawing sperm motility of
Vietnamese native buffalo bulls was similar to that of Murrah bulls
(48.1%) reported by Singh (2010).
Table 3.29. Post thawing sperm motility of Vietnamese native
buffalo bulls
Bull no.
Standardized
Post thawing sperm motility
collection
(%)
(times)
Mean
SD
301
87
49.42ab
0.81
302
81
48.06b
0.74
304
101
49.10ab
0.70
305
108
52.03a
0.89
21
306
75
47.24b
0.97
307
65
47.78b
0.85
Average
517
49.17
0.84
Notes: Values within a column bearing different superscript differ
significantly (P<0.05).
3.5.2. Conception rate of buffalo cows inseminated with semen
straw of Vietnamese native buffalo bulls.
Results of conception rate of buffalo cows in the first
insemination using Vietnamese frozen semen straws in this study
varied from 44.00 to 56.00% (P>0.05). These results were similar to
that of India buffalo (51.84%) reported by Gokhale and Bhagat
(2000).
According to Vale (1997), conception rate of more than 50%
for the first insemination was considered as a sucessful result in
buffalo artificial insemination. Thus, conception rate of buffalo cows
in the first insemination using Vietnamese frozen semen straws in
this study was good.
Bảng 3.31. Conception rate of buffalo cows inseminated with
semen straw of Vietnamese native buffalo bulls.
Total buffalo cows
Pregnant
inseminated
buffalo cows
301
25
13
52.00
302
25
12
48,00
304
25
13
52.00
305
25
14
56.00
Bull no.
Conception rate (%)
22
306
25
11
44.00
307
25
13
52.00
Total
150
76
50.67
CONCLUSIONS AND RECOMMENDATIONS
1. CONCLUSIONS
- Vietnam buffalo bulls in this research had a high mating
reflect for semen collection by an artificial vagina. Age at the first
semen collection of buffalo bulls was 29.83 months old with a body
weight of 518.50 kg.
- Average semen volume, sperm motility and sperm
concentration of Vietnamese native buffalo bulls was 3.89ml,
75.08% and 1.14 x 109 cells/ml, respectively. Individual and season
had effects on some characteristics of semen quality and quantity
(P<0.05). Quality of semen in the summer was the lowest, followed
by quality of semen in the spring. The highest quality of semen was
found in the winter. The quality and quantity of Vietnamese native
buffalo bulls were qualified for production of frozen semen.
- Extender containing 1.363g Tris, 0.762g axit Citric, 0.375g
Fructose, 1.5g Lactose, 2.7g Raffinose, 100,000 UI Penicillin G,
100mg Streptomycin, 6.5% glycerin, 20% egg yolk and distilled
water was good for dilution and storage of frozen semen of
Vietnamese native buffalo bulls. The slow freezing method gave the
best quality of frozen semen straw produced.
- Percentage of semen collection meeting standards for
volume and sperm concentration and sperm motility was 100, 100
and 71.81%, respectively. Vietnamese native buffalo bulls had a
good potential for production of standardized semen straws. On
23
average, number of standardized semen straws was 152.94
straw/colletion/buffalo bull. Individual and season affected frozen
semen straws produced in one collection (P<0.05).
- Quality of frozen semen straw of Vietnamese native buffalo
bulls was good, post thawing sperm motility was 49.17%. The
conception rate in the first insemination of local buffalo cows was
50.67% and varied from 44.00% to 56.00%. No difference in
conception rate among individual buffalo bulls was recorded.
Semen quantity and quality of 06 Vietnamese buffalo bulls
tested were good. Their semen coul be used for production of frozen
semen straws in artificial insemination programs.
2. RECOMMENDATION
- Extender Est3 and method PP2 would be used for
production of frozen semen straws of Vietnamese native buffalo bulls.
All 6 Vietnamese native buffalo bulls tested in this study would be
used for production of frozen semen straws of Vietnamese native
buffalo bulls.
- Further study on effects of other factors such as: age, feed,
sperm collection management, storage time of semen e.t.c. on semen
quantity and quality of Vietnamese native buffalo bulls needs to be
undertaken.
Further study on improvement of native buffalo conception
rate when frozen semen straws was used, needs to be widely
conducted.
24
SCIENTIFIC PUBLICATIONS
Ha Minh Tuan, Mai Van Sanh, Le Van Thong, Le Ba Que. 2014.
Effect of different individual and season on semen quantity
and quality of native buffalo bulls (Swamp buffalo). Journal of
Animal Science and Technology. 48: 68-75.
Ha Minh Tuan, Mai Van Sanh, Le Van Thong, Le Ba Que. 2014.
Effect of dilution extender and freezing method on frozen
semen quality of swamp buffalo. Journal of Animal Husbandry
Sciences and Technics. 6(183): 65-76.
Ha Minh Tuan, Mai Van Sanh, Le Van Thong, Le Ba Que. 2014.
Frozen semen productivity of Vietnamese native buffalo
(swamp buffalo) bulls. Journal of Animal Husbandry Sciences
and Technics. 10(187): 76-82.