Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa

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Reprod Dom Anim 44, 552–569 (2009); doi: 10.1111/j.1439-0531.2008.01240.x
ISSN 0936-6768

Review Article
Factors Aﬀecting the Quality of Cryopreserved Buﬀalo (Bubalus bubalis) Bull
Spermatozoa
SMH Andrabi
Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan

Contents
Storage of buﬀalo (Bubalus bubalis) bull semen in the
cryopreserved state is discussed in this article. Fertility rate
in buﬀalo following artiﬁcial insemination with frozen–thawed
semen is reviewed. To better understand the freezability of
bubaline spermatozoa, the available data on biochemical
components and the activity of speciﬁc enzymes of
semen ⁄ spermatozoa are given. Moreover, the major factors
that may inﬂuence the post-thaw viability and fertility of
buﬀalo spermatozoa are examined in detail. In addition,
suggestions for improvement in cryogenic procedures for
buﬀalo spermatozoa are also given.

Introduction
The domestic buﬀalo, Bubalus bubalis, is a distinct
species within the Bovidae family. The buﬀalo population is continuously increasing, and is estimated at over
170 million head (Food and Agricultural Organization
(FAO) 2004). More than 95% of the population is
located in Asia, where buﬀaloes play a prominent role in
rural livestock production providing the milk, meat and
work draft force. In recent decades, buﬀalo farming has
also expanded widely in Mediterranean areas and in

Latin America.
Only in India and Pakistan are there well-deﬁned
buﬀalo breeds (Drost 2007). There are 18 river buﬀalo
breeds in South Asia, which are further classiﬁed into
ﬁve major groups designated as the Murrah, Gujarat,
Uttar Pradesh, Central Indian and South Indian breeds.
The Nili-Ravi buﬀalo, belonging to the Murrah group,
is recognized as the highest milk-producing breeds of
buﬀalo (Cockrill 1974). The swamp buﬀalo found in
Southeast and Far East Asia has low milk production,
and is mostly used as a draft animal by small farm
holder or is utilized for meat purpose.
The production potential of livestock can be increased
by genetic improvement using one of the modern ways of
breed improvement, e.g., artiﬁcial insemination (AI).
Moreover, the quality of frozen–thawed semen is one of
the most inﬂuential factors aﬀecting the likelihood of
conception (Saacke 1984). Application of AI with frozen–
thawed semen has been reported on a limited scale in
buﬀalo, because of poor freezability and fertility of
buﬀalo spermatozoa when compared with cattle spermatozoa (Kakar and Anand 1981; Muer et al. 1988; Raizada
et al. 1990; Singh and Pant 2000; Andrabi et al. 2001,
2008; Ahmad et al. 2003; Senatore et al. 2004; Kumaresan et al. 2005). Hence, successful cryopreservation of
bubaline semen would aid in the creation of long-term

storage of male gametes and the maintenance of genetic
stock that could improve milk and beef production and its
associated economic value internationally.
This article deals with the storage of bubaline
spermatozoa in deep-frozen ()196°C) state and reviews

the major factors aﬀecting the viability and fertility of
cryopreserved buﬀalo spermatozoa.

Cryopreservation of Spermatozoa
Cryopreservation is a non-physiological method that
involves a high level of adaptation of biological cells to
the osmotic and thermic shocks that occur both during
the dilution, cooling–freezing and during the thawing
procedures (Watson et al. 1992; Holt 2000a,b). Damage
occurring during the freezing–thawing procedures aﬀect
mainly cellular membranes (plasma and mitochondrial)
and in the worst case, the nucleus (Blesbois 2007). This
damage to membranes has consequences on viability
and diﬀerent metabolic factors including adenosine
triphosphate (ATP) concentration in spermatozoa.
Therefore, such changes in the integrity of spermatozoa
aﬀect the viability and fertility. Table 1 summarizes
diﬀerent stresses encountered by the cell and the eﬀect
on the cell of each stressor during the cryogenic
processes.
The ﬁrst successful freezing of buﬀalo semen was
reported by Roy et al. (1956). Basirov (1964) was the
ﬁrst to report the pregnancy with frozen–thawed buﬀalo
bull spermatozoa. Since then, AI has been adopted in
buﬀaloes; however, it remains unpopular because of
poor fertility rate with frozen–thawed semen (Muer
et al. 1988; Andrabi et al. 2001; Ahmad et al. 2003;
Senatore et al. 2004; Kumaresan et al. 2005, 2006;
Shukla and Misra 2007).
A summary of available studies on fertility of frozen

buﬀalo spermatozoa with AI is presented in Table 2. A
critical assessment in term of ﬁrst service conception rate
of the reports given in Table 2 is diﬃcult, as in most of
the studies; the number of inseminations was low.
Details such as number of spermatozoa per dose of AI
and freezing protocol were not provided for some of the
studies. Few studies even lacked the basic information,
like on the type of extender used for cryopreservation or
the total number of animals inseminated. However,
despite the shortcomings in the published reports
(Table 2), it can be suggested that the pregnancy rate
in buﬀalo with AI using frozen–thawed semen is not
comparable with that of cattle.

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa
Table 1. Sources of injury from freeze-thawing of cells (Morris and
Clarke 1987)
Stress encountered
Temperature reduction
Increased solute concentration
Increased ionic concentration
Dehydration
Precipitation of salts and
eutectic formation
Gas bubble formation
Increased solution viscosity
Changes in pH

Direct contact between cells

Potential cellular response
Membrane lipid phase changes
and depolymerization of the cytoskeleton
Osmotic shrinkage
Direct eﬀects on membranes, including
solubilization of membrane proteins
Destabilization of the lipid bilayers
Unknown
Mechanical damage to membranes
and the cytoskeleton
Possible limitation of diﬀusion processes
Denaturation of proteins
Membrane damage

Conception rate in buﬀaloes inseminated with frozen–
thawed semen under ﬁeld condition is approximately
30% (Chohan et al. 1992; Anzar et al. 2003). Published
reliable studies on the fertility of liquid stored buﬀalo
semen seem not to be available (Sansone et al. 2000).
However, few scattered reports indicate a pregnancy
rate of approximately 60% with liquid semen AI in
buﬀaloes (Tomar and Singh 1970; Akhter et al. 2007).
Therefore, a pregnancy rate higher than 50% is regarded
as a good result after AI with frozen–thawed spermatozoa in buﬀalo (Vale 1997). It is relevant to mention
that the same pregnancy rates i.e., near 50% under
normal circumstances are considered poor in cattle with
frozen–thawed spermatozoa.
From above, it is suggested that cryopreservation

adversely aﬀects the viability and the fertilizing potential
of buﬀalo bull spermatozoa. Therefore, there is a need
to discuss in depth the major factors inﬂuencing the
successful cryopreservation of buﬀalo spermatozoa.

Factors Affecting Freezability
Biochemical characteristics of semen
It is reported that buﬀalo spermatozoa are more susceptible to hazards during freezing and thawing than cattle
spermatozoa, thus resulting in lower fertilizing potential
(Raizada et al. 1990; Andrabi et al. 2008). Moreover,
there are speciﬁc biochemical factors that aﬀect the ability
of spermatozoa to prevent damages caused by the
cryogenic procedures. One of the many possible causes
of lower freezability of buﬀalo bull semen compared to
cattle bull can be due to the diﬀerences in the lipid ratio of
the spermatozoa (Jain and Anand 1976; Tatham 2000;
European Regional Focal Point on Animal Genetic
Resources, 2003). For example, phosphatidyl choline
makes up approximately 66% of all phospholipids found
in buﬀalo sperm plasma membrane (Cheshmedjieva and
Dimov 1994) but approximately 50% in case of cattle bull
sperm membrane (Parks et al. 1987). Similarly, phosphatidyl ethanolamine makes up approximately 23% of all
phospholipids present in buﬀalo sperm plasmalemma
(Cheshmedjieva and Dimov 1994) but almost 10% in case
of cattle bull sperm membrane (Parks et al. 1987).
To better, understand the nature of bubaline spermatozoa the available data on biochemical components
and the activity of speciﬁc enzymes of semen are given in

553

Tables 3 and 4. The values of diﬀerent constituents
given in Tables 3 and 4 show that buﬀalo whole
semen ⁄ seminal plasma ⁄ spermatozoa ⁄ plasma membrane
compared to cattle have distinct characteristics, particularly the membrane lipid ratio. Therefore, there is a
need to develop biochemically deﬁned extenders and
cryogenic procedures that are species speciﬁc, and may
result in the improvement of viability and fertility of
frozen–thawed buﬀalo spermatozoa.
Buffer
Dilution of semen in a suitable buﬀer is one of the
important factors aﬀecting sperm survival during cryopreservation (Rasul et al. 2000). An ideal buﬀer should
have: (i) pH between 6 and 8, preferably 7; (ii) maximum
water solubility and minimum solubility in all other
solvents; (iii) minimum salt eﬀects; (iv) minimum buﬀer
concentration; (v) least temperature eﬀect; (vi) wellbehaved cation interactions; (vii) greater ionic strengths
and (viii) chemical stability (Bates 1961; Good et al.
1966; Good and Izawa 1972; Keith and Morrison 1981).
Development of a suitable buﬀering system for the
cryopreservation of buﬀalo spermatozoa has been in
progress for sometime (Rasul et al. 2000). Several
studies have concentrated on the use of chemically
deﬁned buﬀers for buﬀalo semen. In this regard,
Matharoo and Singh (1980) tested citrate, Tris or citric
acid as buﬀers for deep-freezing of buﬀalo spermatozoa.
They found that freezing loss was least with Tris-based
extender as judged by post-thaw motility. Similarly,
Chinnaiya and Ganguli (1980a) found better post-thaw
sperm motility with Tris-based extender than citrate or
citric acid-based extenders. In another study, Chinnaiya
and Ganguli (1980b) found that spermatozoon frozen in

citric acid, citrate or Tris-based extender showed similar
degree of acrosomal damage and similar recovery rates.
However, acrosin activity was greatest in citrate-based
diluent and least in Tris buﬀer.
Ahmad et al. (1986) found that Tris–citric acid based
extender is suitable for the cryopreservation of buﬀalo
spermatozoa in terms of post-thaw motility and survivability. Later on, Dhami and Kodagali (1990) studied
the eﬀects of semen extenders based on Tris or citrate
buﬀer. It was reported that Tris-based extender
improved the freezability of buﬀalo spermatozoa as
judged by the extracellular release of spermatozoal
enzymes and in vivo fertility. Similarly, Singh et al.
(1990, 1991) studied semen diluents based on citrate or
Tris or citric acid for freezing of buﬀalo spermatozoa.
They found that with Tris-based extender there was least
release of lactic dehydrogenase and sorbitol dehydrogenase in buﬀalo spermatozoa during cryopreservation
followed by citrate and citric acid-based extenders. In
addition, Tris provided the highest protection against
acrosomal damage compared to other buﬀers tested.
Dhami et al. (1994) studied the eﬀects of semen
extenders based on Tris or citrate. It was found that
Tris-based extender yielded higher post-thaw spermatozoal motility. Singh et al. (2000) compared Tris-buﬀer
with Laiciphos (IMV, L’Aigle, France; containing
laiciphos, egg yolk, distilled water and unknown buﬀer)
and Biociphos (IMV, France; containing biociphos with

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

554

SMH Andrabi

Table 2. Fertility rate in buﬀalo following AI with frozen–thawed semen
Reference
Bhosrekar and Nagarcenkar 1971
Bandyopadhyay and Roy 1975
Chinnaiya et al. 1979
Singh et al. 1980
Tuli et al. 1981a
Matharoo and Garcha 1986
Heuer and Bajwa 1986
Heuer et al. 1987

Singh and Singh 1988
Ahmad et al. 1988
Bhavsar et al. 1988
Bhavsar et al. 1989a
Bhavsar et al. 1989b

Singh 1990
Haranath et al. 1990
Dhami and Kodagali 1990

Dhami and Kodagali 1991
Hassan and Zia Ur 1994
Dhami et al. 1994

Barnabe et al. 1994
Dhami et al. 1996

Younis et al. 1999
Barile et al. 1999b
Gokhale and Bhagat 2000
Sukhato et al. 2001c
Pant et al. 2001
Prabhakar et al. 2002
Taraphder et al. 2003
Sosa et al. 2003
Presicce et al. 2004d
Kanchan and Singh 2005
Anzar et al. 2003
Andrabi et al. 2006

Extender

Total number of ﬁrst AI

Skim milk powder–yolk–glycine–citrate–
fructose–glycerol
Yolk–citrate–glycerol
Yolk–citrate–glycerol, Citric
acid–whey–glycerol and Tris–yolk–glycerol
Tris–yolk–glycerol
Tris–yolk–glycerol
Tris–yolk–glycerol
Information not provided
Lactose–fructose–yolk–glycerol, Skim
milk–fructose–yolk–glycerol and
Tris–fructose–yolk–glycerol
Information not available

Yolk–glycerol with milk or lactose or
fructose and lactose
Yolk–lactose–fructose–glycerol
Tris–fructose–yolk–glycerol
Tris–fructose–yolk–glycerol with or without
additives (L-cysteine HCl H2O, sheep
hyaluronidase, beta-amylase or acetylcholine
chloride)
Information not provided
Tris–egg yolk–glycerol
Tris–fructose–yolk–glycerol,
Yolk–citrate–glycerol and Lactose–yolk
glycerol
Information not provided
Information not provided
Tris–yolk–glycerol–, Citrate–yolk–glycerol– and
Lactose–yolk–glycerol–, with or without
(control) cysteine, EDTA and raﬃnose
Tris–TES and Tris–yolk
Tris–citric acid–fructose–yolk–glycerol
and Whole cow’s milk–yolk–glycerol
Lactose–fructose–yolk–glycerol
Information not available
Information not provided
Tris–fructose–yolk–glycerol
Information not provided
Information not available
Information not provided
Milk-, Laiciphos- and Tris- with or without
glycerol, DMSO and propylene glycol

Information not available
Information not available
Information not available
Tris–citric acid–fructose–yolk–glycerol

Over all ﬁrst service aCR (%)

109

45.0

Information not available
315

40.6
53.93

72
159
825
61 952
3220

45.8
35.22
39.30
51.78
37.4

218

2745

41.0
44.7

1908
Information not available
3791

39.2
45.85
46.0

Information not provided
Information not available
3412

39.7
51.53
39.9

2995
1110
853

40.1
65.26
57.95

Information not available

806

53.14
63.98

971
217
6762
178
202
1941
Information not available
Information not available

41.8
42.5
52.0
37.0
34.95
59.15
40.75
50.6

67
Information not available
Information not available
432

48.0
29.87

29.0
53.0

a

Pregnancies were conﬁrmed through rectal palpations.
Buﬀalo were synchronized with a progesterone-releasing intravagiral device (PRID) containing progesterone and oestradiol benzoate, for 10 days. Seven days after
insertion of PRID the buﬀalo received an injection of pregnant mare serum gonadotropin (PMSG) and an injection of cloprostenol. AI was performed 48, 72 or 96 h
after removal of the device.
c
Oestrus synchronization was performed by inserting a progesterone-impregnated silicone elastomer device (CIDR-BÒ) into the vagina. Each buﬀalo was injected
intramuscularly with 1 mg of oestradiol benzoate (CIDIROLÒ) on the day of CIDR-B insertion and 150 IU of ECG upon CIDR-B removal (12 days after insertion).
AI was performed between 48 and 50 h after the CIDR-B was removed.
d
AI was performed twice at 72 and 96 h after administration of prostaglandins to buﬀaloes bearing a functional corpus luteum.
b

glycerol, egg yolk, distilled water and unknown buﬀer)
for cryopreservation of buﬀalo semen. Again they found
that Tris-based extender was better compared to Laiciphos and Biociphos as judged by post-thaw motility and
survivability.
Rasul et al. (2000) carried out a study to identify the
suitable buﬀer for cryopreservation of buﬀalo semen.
The buﬀers tested were tri-sodium citrate, Tris–citric
acid, Tris–Tes or Tris–Hepes. They found that Tris–
citrate tended to be better in term of improving the postthaw motion characteristics of buﬀalo spermatozoa.
Nonetheless, plasma membrane integrity and normal
acrosomes of spermatozoa did not vary because of

buﬀering systems. Conversely, Oba et al. (1994) and

Chachur et al. (1997) found that Tes is to be equal value
to Tris-based extender in terms of post-thaw motility,
acrosome retention or membrane integrity.
From the results of the above mentioned studies, it is
suggested that zwitterion buﬀers particularly, Tris–citric
acid may provide the most satisfactory buﬀering system
to improve the post-thaw freezability and consequently
may also improve the fertility of buﬀalo spermatozoa. It
is believed that zwitterion buﬀers have pH nearer to the
pKa (acid dissociation constant). Also there pKa is least
inﬂuenced by temperature as compared to other buﬀers
(Graham et al. 1972).

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa

555

Table 3. Biochemical composition of buﬀalo semen
Characteristic of
component

Reference

Whole semen

Seminal plasma

Spermatozoa

Rattan et al. 1980

23.47 mg ⁄ 100 ml

Dabas et al. 1984

82 ± 6 mg ⁄ 100 ml

167 ± 9
lg ⁄ 1011 cells

0.024 ± 0.003
lmol ⁄ ml
3.9 ± 0.5
mg ⁄ 100 ml

0.066 ± 0.014
lmol ⁄ 109cells

Banerjee and Ganguli
1973

0.091 ± 0.011
lmol ⁄ ml
6.2 ± 0.8
mg ⁄ 100 ml

Citric acid

Banerjee and Ganguli
1973

441.8 ± 31.9
mg ⁄ 100 ml

444.9 ± 17.4
mg ⁄ 100 ml

Fructose

Salem and Osman 1972

Lactic acid

Ascorbic acid

Jain 1987

Banerjee and Ganguli
1973
Rattan et al. 1980

Lipids

368.12–
430.92 mg ⁄ 100 ml

815.71 mg ⁄ 100 ml

1.500 mg ⁄ ml

1.147 mg ⁄ 109 cells

Sarmah et al. 1983

1.750 ± 0.030
mg ⁄ ml

1.320 ± 0.030
mg ⁄ 109 cells

Cholesterol

Mohan et al. 1979

Phospholipids

Jain and Anand 1976

0.594 mg ⁄ ml

0.548 mg ⁄ 109 cells

Sidhu and Guraya 1979

0.1735 ± 0.0256
mg ⁄ ml
0.069 ± 0.02

mg ⁄ ml

0.3074 ± 0.0923
mg ⁄ 109 cells
0.064 ± 0.02
mg ⁄ 109 cells

Jain and Anand 1976

21.7 ± 1.0% of
total
phospholipids

30.4 ± 1.4% of
total
phospholipids

Sarmah et al. 1983

34.1 ± 1.8% of
total
phospholipids

28.0 ± 1.2% of
total
phospholipids

Jain and Anand 1976

17.3 ± 0.9% of

total
phospholipids

19.4 ± 1.7% of
total
phospholipids

Phosphatidyl choline

Phosphatidal
choline (choline
plasmogen)

Amount of lactic acid in cattle bull seminal
plasma is 72 ± 5 mg ⁄ 100 ml (Dabas et al.
1984)
Amount of lactic acid in cattle bull
spermatozoa is 352 ± 16 lg ⁄ 1011 cells
(Dabas et al. 1984)
Amount of ascorbic acid in whole semen,
seminal plasma and spermatozoa of cattle
bull is 0.131 ± 0.030,
0.505 ± 0.0185 lmol ⁄ ml
and 0.0832 ± 0.0337 lmol ⁄ 109cells,
respectively (Jain 1987)
Amount of citric acid in whole semen and
seminal plasma of cattle bull is
531.3 ± 73.4 and 576.9 ± 58.6 mg ⁄ 100
ml, respectively (Banerjee and Ganguli
1973)

Amount of fructose in seminal plasma of
cattle bull is 519.07–618.93 mg ⁄ 100 ml
(Salem and Osman 1972)

623.8 ± 83.6
mg ⁄ 100 ml

Jain and Anand 1976

Sarmah et al. 1983

Comment

91.84 ± 3.91–
141.88 ± 3.12
mg ⁄ 100 ml

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Amount of fructose in whole semen of cattle
bull is 780.6 ± 66.2 mg ⁄ 100 ml (Banerjee
and Ganguli 1973)
Amount of lipids in seminal plasma and
spermatozoa of cattle bull is 2.900 mg ⁄ ml
and 0.703 mg ⁄ 109 cells, respectively (Jain
and Anand 1976)
Amount of lipids in seminal plasma and
spermatozoa of cattle bull is
1.04 ± 0.2 mg ⁄ ml and 2.18 ± 0.22
mg ⁄ 109 cells, respectively (Pursel and

Graham 1967)
Amount of cholesterol in whole semen of
cattle bull is 104–412 mg ⁄ 100 ml
(RoyChoudhury 1970)
Amount of phospholipids in seminal plasma
of cattle bull is 1.491 mg ⁄ ml (Jain and
Anand 1976)

Amount of phospholipids in spermatozoa of
cattle bull is 0.416 mg ⁄ 109 cells (Jain and
Anand 1976)
Amount of phosphatidyl choline in seminal
plasma of cattle bull, which according to
Pursel and Graham (1967), Clegg and
Foote (1973), and Jain and Anand (1976) is
30.0, 26.3 and 24.5 ± 2.2% of total phospholipids, respectively
Amount of phosphatidayl choline in
spermatozoa of cattle bull, which
according to Pursel and Graham (1967),
Clegg and Foote (1973), and Jain and
Anand (1976) is 35.6, 30.1 and
17.9 ± 0.8% of total phospholipids,
respectively
Amount of phosphatidayl choline in semi
nal plasma of cattle bull, which according
to Pursel and Graham (1967), Clegg and
Foote (1973), and Jain and Anand (1976) is
23.6%, 17.6% and 32.9 ± 2.0% of total
phospholipids, respectively
Amount of phosphatidayl choline in

spermatozoa obtained from cattle bull,
which according to Pursel and Graham
(1967), Clegg and Foote (1973), and Jain
and Anand (1976) is 28.0%, 31.8% and
36.8 ± 1.4% of total phospholipids,
respectively

556

SMH Andrabi

Table 3. Continued
Characteristic of
component
Phosphatidyl
ethanolamine

Reference

Whole semen

Seminal plasma

Spermatozoa

Jain and Anand
1976

11.7 ± 1.5% of

total
phospholipids

10.8 ± 2.0% of
total
phospholipids

Sarmah et al.
1983

10.8 ± 1.4% of
total
phospholipids

9.3 ± 1.2% of total
phospholipids

Jain and Anand
1976

4.1 ± 0.3% of total
phospholipids

3.4 ± 0.5% of total
phospholipids

Sarmah et al.
1983

4.9 ± 0.7% of total

phospholipids

5.7 ± 0.7% of total
phospholipids

Jain and Anand
1976

13.1 ± 0.7% of
total
phospholipids

11.3 ± 0.7% of
total
phospholipids

Sarmah et al.
1983

13.8 ± 0.8% of
total
phospholipids

17.4 ± 1.3% of
total
phospholipids

Phosphatidyl serine

Jain and Anand

1976

2.8 ± 0.4% of total
phospholipids

1.5 ± 0.3% of total
phospholipids

Phosphatidyl
serine +
phosphatidyl
inositol

Sarmah et al.
1983

6.1 ± 0.7% of total
phospholipids

8.1 ± 0.3% of total
phospholipids

Phosphatidyl
inositol

Jain and Anand
1976

2.9 ± 0.5% of total
phospholipids

0.6 ± 0.1% of total
phospholipids

Lysophosphatidyl
choline

Jain and Anand
1976

3.9 ± 0.9% of total
phospholipids

3.9 ± 0.5% of total
phospholipids

Sarmah et al.
1983

3.1 ± 0.2% of total
phospholipids

8.3 ± 0.1% of total
phospholipids

Phosphatidal
ethanolamine
(ethanolamine
plasmogen)

Sphinogomyelin

Comment
Amount of phosphatidyl ethanolamine in
seminal plasma of cattle bull, which
according to Pursel and Graham (1967),
Clegg and Foote (1973), and Jain and
Anand (1976) is 10.5, 5.4 and 5.6 ± 0.4%
of total phospholipids, respectively
Amount of phosphatidyl ethanolamine in
spermatozoa of cattle bull which according
to Pursel and Graham (1967), Clegg and
Foote (1973), and Jain and Anand (1976) is
20.0%, 9.7% and 5.3 ± 0.4% of total
phospholipids, respectively
Amount of phosphatidal ethanolamine in
seminal plasma of cattle bull, which
according to Pursel and Graham (1967),
Clegg and Foote (1973), and Jain and
Anand (1976) is16.3%, 5.0% and
9.0 ± 0.9% of total phospholipids,
respectively
Amount of phosphatidal ethanolamine in
spermatozoa of cattle bull, which according to Pursel and Graham (1967), Clegg
and Foote (1973), and Jain and Anand
(1976) is 7.2%, 4.1% and 9.0 ± 0.4% of
total phospholipids, respectively
Amount of sphinogomyelin in seminal
plasma of cattle bull which according to
Pursel and Graham (1967), Clegg and

Foote (1973), and Jain and Anand (1976)
is16.3%, 13.2% and 11.6 ± 1.0 9% of
total phospholipids, respectively
Amount of sphinogomyelin in cattle bull
spermatozoa which according to Pursel
and Graham (1967), Clegg and Foote
(1973), and Jain and Anand (1976) is 9.1%,
11.5% and 12.2 ± 1.2% of total phospholipids, respectively
Amount of phosphatidyl serine in seminal
plasma of cattle bull is 1.3 ± 0.3% of total
phospholipids (Jain and Anand 1976)
Amount of phosphatidyl serine in
spermatozoa of cattle bull is 1.7 ± 0.4%
of total phospholipids (Jain and Anand
1976)
Amount of Phosphatidyl serine +
Phosphatidyl inositol in seminal plasma of
cattle bull is 3.6% of total phospholipids
(Clegg and Foote 1973)
Amount of phosphatidyl serine +
phosphatidyl inositol in spermatozoa of
cattle bull is 0.7% of total phospholipids
(Clegg and Foote 1973)
The value of phosphatidyl inositol in
seminal plasma obtained in this study
diﬀer from that of cattle bull which
according to Jain and Anand (1976) is
0.8 ± 0.2% of total phospholipids
The value of phosphatidyl inositol in
spermatozoa obtained in this study diﬀer

from that of cattle bull which according to
Jain and Anand (1976) is 1.0 ± 0.2% of
total phospholipids
Amount of lysophosphatidyl choline in
seminal plasma of cattle bull, which
according to Clegg and Foote (1973), and
Jain and Anand (1976) is 2.2% and
1.2 ± 0.3% of total phospholipids,
respectively
Amount of lysophosphatidyl choline in
spermatozoa of cattle bull which according
to Clegg and Foote (1973), and Jain and
Anand (1976) is 1.7% and 1.9 ± 0.5% of
total phospholipids, respectively

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa

557

Table 3. Continued
Characteristic of
component

Reference

Lysophosphatidyl
ethanolamine

Whole semen

Seminal plasma

Spermatozoa

Comment

Jain and Anand
1976

5.6 ± 1.5% of total
phospholipids

4.4 ± 1.0% of total
phospholipids

Sarmah et al.
1983

6.6 ± 1.0% of total
phospholipids

Lysophosphatidyl
serine

Jain and Anand
1976

1.0 ± 0.3% of total
phospholipids

0.7 ± 0.1% of total
phospholipids

Diphosphatidyl
glycerol
(cardiolipin)

Jain and Anand
1976

7.4 ± 1.3% of total
phospholipids

5.5 ± 0.7% of total
phospholipids

Sarmah et al.
1983

3.5 ± 0.5% of total
phospholipids

4.9 ± 0.4% of total
phospholipids

Phosphatidic acid

Jain and Anand
1976

0.5 ± 0.2% of total
phospholipids

0.3 ± 0.1% of total
phospholipids

Neutral lipids

Jain and Anand
1976

0.439 mg ⁄ ml

0.286 mg ⁄ 109 cells

Glycolipids

Jain and Anand
1976

0.581 mg ⁄ ml

0.397 mg ⁄ 109 cells

Glutathione

Jain et al. 1990

Aspartic acid

Chaudhary and
Gangwar 1977

0.395 mM

Glutamic acid

Chaudhary and
Gangwar 1977

4.28 mM

Serine

Chaudhary and
Gangwar 1977

0.60 mM

Alanine

Chaudhary and
Gangwar 1977

0.413 mM

Glycine

Chaudhary and
Gangwar 1977

1.34 mM

Lysine

Chaudhary and
Gangwar 1977

0.133 mM

Amount of lysophosphatidyl ethanolamine
in seminal plasma of cattle bull, which
according to Clegg and Foote (1973), and
Jain and Anand (1976) is 2.2% and
1.2 ± 0.3% of total phospholipids,
respectively
Amount of lysophosphatidyl ethanolamine
in spermatozoa of cattle bull which
according to Jain and Anand (1976) is
3.2 ± 0.6% of total phospholipids
Amount of lysophosphatidyl serine in
seminal plasma of cattle bull is
0.4 ± 0.1% of total phospholipids (Jain
and Anand 1976)
Amount of lysophosphatidyl serine in
spermatozoa of cattle bull is 0.5 ± 0.1%
of total phospholipids (Jain and Anand

1976)
Amount of cardiolipin in seminal plasma of
cattle bull, which according to Pursel and
Graham (1967), Clegg and Foote (1973),
and Jain and Anand (1976) is 5.4%, 8.8%
and 5.0 ± 0.5% of total phospholipids,
respectively
Amount of cardiolipin in spermatozoa of
cattle bull, which according to Clegg and
Foote (1973), and Jain and Anand (1976) is
6.3% and 5.9 ± 1.0% of total phospholipids, respectively
Amount of phosphatidic acid in seminal
plasma of cattle bull is 0.4 ± 0.1% of total
phospholipids (Jain and Anand 1976)
Amount of phosphatidic acid in
spermatozoa obtained of cattle bull is
0.2 ± 0.1% of total phospholipids (Jain
and Anand 1976)
Amount of neutral lipids in seminal plasma
of cattle bull is 0.896 mg ⁄ ml (Jain and
Anand 1976)
Amount of neutral lipids in spermatozoa of
cattle bull is 0.164 mg ⁄ 109 cells (Jain and
Anand 1976)
Amount of glycolipids in seminal plasma of
cattle bull is 0.713 mg ⁄ ml (Jain and Anand
1976)
Amount of glycolipids in spermatozoa of
cattle bull is 0.154 mg ⁄ 109 cells (Jain and
Anand 1976)

Amount of glutathione obtained in whole
semen of cattle bull is
45.35 ± 5.07 lmol ⁄ ml (Jain and Anand
1976)
Amount of aspartic acid in seminal plasma
of cattle bull is 0.369 ± 0.025 lmoles ⁄ ml
(Al-Hakim et al. 1970)
Amount of glutamic acid in seminal plasma
of cattle bull is 4.352 ± 0.257 lmoles ⁄ ml
(Al-Hakim et al. 1970)
Amount of serine in seminal plasma of
cattle bull is 0.506 ± 0.03 lmoles ⁄ ml
(Al-Hakim et al. 1970)
Amount of alanine in seminal plasma of
cattle is 1.078 ± 0.071 lmoles ⁄ ml (AlHakim et al. 1970)
Amount of glycine in seminal plasma of
cattle bull is 0.564 ± 0.031 lmoles ⁄ ml
(Al-Hakim et al. 1970)
Amount of lysine in seminal plasma of cattle
bull is 0.177 ± 0.010 lmoles ⁄ ml (Al-Hakim et al. 1970)

32.49 ± 5.10
lmol ⁄ ml

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

558

SMH Andrabi

Table 3. Continued
Characteristic of
component

Reference

Deoxyribo-nuclease

Chauhan et al. 1975

Acid phosphatase

Chauhan and
Srivastava 1973
Dabas et al. 1984

Alkaline
phosphatase

Chauhan and
Srivastava 1973
Dabas et al. 1984

Whole semen

Seminal plasma

Spermatozoa
2007.33 ± 112.01

KU ⁄ ml

315.31 ± 22.66
BU ⁄ 100 ml
194 ± 10
BU ⁄ 100 ml
312.50 ± 24.04
BU ⁄ 100 ml
270 ± 9
BU ⁄ 100 ml

39 ± 6 BU ⁄ 1011
cell

63 ± 6 BU ⁄ 1011
cell

Comment
Amount of deoxyribonuclease in
spermatozoa of cattle bull is
1843.4 ± 126.36 KU ⁄ ml (Chauhan et al.
1975)
Amount of acid phosphatase in seminal
plasma and spermatozoa of cattle bull is
182 ± 10 BU ⁄ 100 ml and 25 ± 2 BU ⁄
1011 cell respectively (Dabas et al. 1984)
Amount of alkaline phosphatase in seminal
plasma and spermatozoa of cattle bull is
246 ± 8 BU ⁄ 100 ml and
54 ± 3 BU ⁄ 1011 cell, respectively (Dabas

et al. 1984)

a

Values are mean ± SEM.

Table 4. Phospholipid composition (% of total phospholipids) of plasma membrane of buﬀalo and cattle bull spermatozoa
Phospholipid
Phosphatidyl ethanolamine
Phosphatidyl choline
Phosphatidyl serine
Phosphatidyl inositol
Sphinogomyelin
Lysophosphatidyl choline
Phosphatidyl glycerol
Diphosphatidyl glycerol

Buﬀalo (Cheshmedjieva and Dimov 1994)
22.9 ±
66.0 ±
3.5 ±
2.5 ±
8.0 ±
–
4.7 ±
5.0 ±

1.6a
3.5
0.5

0.4
1.9
0.6
0.9

Cattle (Parks et al. 1987)
9.9
50.3
1.1
2.7
12.6
1.8
–
–

a

Values are mean ± SE.

Additionally, the diﬀerences regarding eﬃcacy of
diﬀerent buﬀers suggest that buﬀalo spermatozoa are
more prone to freezing stress as compared to cattle bull
spermatozoa possibly because of biochemical factors
that inﬂuence membrane ﬂuidity during cryogenic preservation (refer to Table 4). Therefore, there is a need to
study the inﬂuence of selected buﬀers on pre- and postcryogenic membrane stability i.e., in terms of biochemical ⁄ molecular level changes in lipid bilayer and phase
transition.
Permeable cryoprotectant
Glycerol is often poly-hydroxylated and capable of
hydrogen bonding with water, capable of permeating
across the cell membrane, and non-toxic during exposure to cells in the concentration between approximately

1–5 mol ⁄ l, depending on cell type and conditions of
exposure (Fuller and Paynter 2004). More speciﬁcally,
the physiological actions of glycerol during the cryopreservation of spermatozoa take place by replacing
intracellular water necessary for the maintenance of
cellular volume, interaction with ions and macromolecules, and depressing the freezing point of water and the
consequent lowering of electrolyte concentrations in the
unfrozen fraction so that less ice forms at any given
temperature (Holt 2000b; Medeiros et al. 2002).
For cryopreservation of buﬀalo semen, several studies
have been carried out in an attempt to ﬁnd the optimum
levels of glycerol and glycerolization. In this context,
Jainudeen and Das (1982) studied the eﬀect of two
glycerolization procedures (one step vs two steps) and

the inﬂuence of glycerol level in the extender (3%, 5% or
7%). They found that glycerolization procedure had no
signiﬁcant eﬀect on sperm survival traits like motility
and acrosomal integrity. They also found that post-thaw
motility of spermatozoa was signiﬁcantly better in a 5%
glycerol extender, whereas the percentage of intact
acrosomes was greater in spermatozoa extended in 3%
or 5% glycerol than in spermatozoa extended in 7%
glycerol.
In another study, Kumar et al. (1992) found that the
best level of glycerol was 6% for Tris- and milk-based
diluents, and 9% glycerol for the sodium citrate diluent
to obtain better post-thaw motility for buﬀalo spermatozoa. Ramakrishnan and Ariﬀ (1994), and Nastri et al.
(1994) also tried to reduce the glycerol concentrations
from 8% to 2% or 3%, but they found that the
reduction in glycerol below 5% decreased the post-thaw

motility and ⁄ or acrosome integrity of spermatozoa in
the extenders tested. Abbas and Andrabi (2002) studied
the eﬀects of diﬀerent concentrations of glycerol (2%,
3%, 4%, 5%, 6%, 7%, 8%, 10% or 12%) on post-thaw
sperm quality. They reported that the spermatozoa
frozen in 7% were signiﬁcantly better to those in other
concentrations of glycerol as judged by post-thaw
motility, survivability and plasma membrane integrity.
Regarding glycerolization, Singh et al. (2006) have
conﬁrmed that single step is more suitable for the
cryopreservation of buﬀalo spermatozoa in terms of
post-thaw forward motility.
Ethylene glycol could be another option for the
cryopreservation of buﬀalo spermatozoa. Permeability
of ethylene glycol was found to be higher than glycerol

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa

in spermatozoa of diﬀerent species (Gilmore et al. 1995,
1998; Phelps et al. 1999), resulting in lower hydraulic
conductivity and then in a reduction in the osmotic
stress to which cells are exposed during cooling and
freezing (Gilmore et al. 1995). Propylene glycol also has
the basic properties of a cryoprotectant i.e., it is miscible
with water in all proportions, its solutions in water have
profoundly depressed freezing points, and presumably,
it has a low intrinsic toxicity as it is widely used in the

food and pharmaceutical industries (Arnaud and Pegg
1990). Recently, Valdez et al. (2003) and Rohilla et al.
(2005) have tested ethylene glycol or propylene glycol as
substitute for glycerol. Their preliminary results suggest
that ethylene glycol may be used for freezing bubaline
spermatozoa. Therefore, there is a need to study in
detail the factors that may aﬀect the viability of frozen
buﬀalo spermatozoa with ethylene glycol as a cryoprotectant. Further studies, are also suggested for testing
propylene glycol as a cryoprotectant for buﬀalo spermatozoa.
Dimethyl sulfoxide (DMSO) is a rapid penetrating
cryoprotectants having lower molecular weight than
glycerol. Also DMSO may inhibit harmful eﬀect of
hydroxyl radicals (Yu and Quinn 1994), as these radicals
appear during cell respiration and are detrimental to cell
(Johnson and Nasr-Esfahani 1994). More recently,
Rasul et al. (2007) studied glycerol and ⁄ or DMSO,
added either at 37°C or at 4°C as a cryoprotectant for
buﬀalo spermatozoa. The concentrations (%) of glycerol and DMSO adjusted were 0 : 0, 0 : 1.5, 0 : 3; 3 : 0,
3 : 1.5, 3 : 3; and 6 : 0, 6 : 1.5, 6 : 3 respectively. It was,
concluded that addition of DMSO at the levels investigated did not improve the post-thaw quality of
spermatozoa. However, glycerol at a concentration of
6%, when added at 37°C, provided the maximum
cryoprotection to the motility apparatus, and plasma
membrane integrity of buﬀalo spermatozoa in Tris–
citric acid based extender. The exact mechanism
involved in the antagonist eﬀect of DMSO on the
cryoprotection ability of glycerol is not understood.
Moreover, the lethal eﬀect of DMSO is attributed to its
toxic eﬀect rather than osmotic (Rasul et al. 2007). It is
believed that because of the lower molecular weight of

DMSO, its penetrating ability into the cell is higher than
glycerol.
From the available studies, it is therefore, suggested
that a glycerol concentration of 5–7% added initially in
the extender may be suitable for the cryopreservation of
buﬀalo bull spermatozoa. On the other hand,
development of less toxic cryoprotectant could make a
signiﬁcant contribution in improving the quality of
frozen–thawed buﬀalo spermatozoa.
Non-permeable cryoprotectant
Egg yolk is a common component of semen freezing
extenders for most of the livestock species, including the
buﬀalo (Sansone et al. 2000). It is widely believed that
low density lipoproteins (LDL) contained in egg yolk is
largely responsible for sperm protection during cryopreservation (Pace and Graham 1974; Watson 1976). It
is suggested that LDL adheres to sperm membrane and
provides protection to sperm by stabilizing the mem-

559

brane. A second hypothesis suggests that phospholipids
present in LDL protect sperm by forming a protective
ﬁlm on the sperm surface or by replacing sperm
membrane phospholipids that are lost or damaged
during the cryopreservation process (Foulkes et al.
1980; Quinn et al. 1980; Graham and Foote 1987). A
third mechanism of protection suggests that LDL seizes
the deleterious proteins present in seminal plasma thus
improving the freezability of spermatozoa (Bergeron
and Manjunath 2006). The exact mechanism by which

EY preserves the spermatozoa during freeze–thaw
process is unknown (Bathgate et al. 2006).
Review of literature reveals that little attention has
been paid to the level of egg yolk necessary for freezing
buﬀalo semen, and generally it is used at a concentration
of 20% in semen extender (Sansone et al. 2000; Andrabi
et al. 2008). Furthermore, the use of egg yolk in higher
concentration may have deleterious eﬀects combined
with toxicity (amino acid oxidase activity) of dead
spermatozoa resulting in lower post-thaw spermatozoal
quality (Shannon 1972). The enhanced toxicity associated with increased egg yolk is probably due to the
elevated substrate available for hydrogen peroxide
formation (Tosic and Walton 1950).
In this regard, Sahni and Mohan (1990) studied
diﬀerent levels of egg yolk in extender as a nonpermeable cryoprotectant for buﬀalo semen. The concentration of egg yolk used was 0%, 2%, 5%, 10% or
20%. They concluded that the concentration of egg yolk
in the extender could be reduced from 20% to 5%
without any compromise in post-thaw motility of
spermatozoa. Kumar et al. (1994) studied the eﬀect of
diﬀerent levels of egg yolk (0%, 1%, 5%, 10% and
20%) in Tris-based extender on sperm motility and
survival before and after freezing in buﬀalo. They found
that the best post-thaw motility and survivability was
with 5% yolk. Singh et al. (1999) studied the eﬀect of
diﬀerent levels of egg yolk on freezability of buﬀalo
semen. They found that egg yolk at 10% was superior
for freezability with regards to pre-freeze and post-thaw
sperm motility. It was, also suggested that 10% egg yolk
is better in a Tris-based extender for freezing buﬀalo
semen compared to at lower concentration (5%).

Recently, Andrabi et al. (2008) investigated the use of
duck egg yolk, Guinea fowl egg yolk and Indian
indigenous hen (Desi) egg yolk in extender for improving the post-thaw quality of buﬀalo bull spermatozoa,
and compared it with commercial hen egg yolk. It was
concluded that duck egg yolk compared to other avian
yolks in extender improves the freezability of buﬀalo
bull spermatozoa as judged by motility, survivability,
plasma membrane integrity, intactness of acrosome and
head, mid-piece and tail abnormalities. In this regard, it
is suggested that the improvement or decline in postthaw quality of mammalian spermatozoa with egg yolk
of diﬀerent avian species in freezing extender is attributed to the diﬀerences in biochemical composition of the
yolks (Trimeche et al. 1997; Bathgate et al. 2006).
Studies investigating the inﬂuence of egg yolk from
diﬀerent avian species on Jackass sperm during freeze–
thawing have found that the ratio of phosphatidyl
ethanolamine : phosphatidyl choline appears to play a
role in the level of protection aﬀorded to the sperm

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

560

SMH Andrabi

(Trimeche et al. 1997). This is of interest to mention that
Bathgate et al. (2006) reported a signiﬁcant diﬀerence in
ratio of phosphatidyl ethanolamine : phosphatidyl choline in chicken egg yolk and duck yolk with a higher
ratio in later. Therefore, it can be put forward that
higher ratio of phosphatidyl ethanolamine : phosphatidyl choline in duck egg yolk may have improved the

freezability of buﬀalo spermatozoa in the study by
Andrabi et al. (2008). It is, also proposed that supplementation of cryodiluent with quail egg yolk for buﬀalo
bull semen needs to be investigated as the ratio of
phosphatidyl ethanolamine : phosphatidyl choline in
quail yolk is even higher than duck yolk as reported
by Bathgate et al. (2006). Finally, as the ﬁndings of
Andrabi et al. (2008) are preliminary, therefore, it is
suggested that further studies are required to establish
the source and levels of egg yolk in freezing medium for
buﬀalo spermatozoa.
Polyethylene glycol (PEG) is a non-permeable cryoprotectant that may slow down the process of ice
nucleation during cryogenic process, thus protecting the
cellular membrane. Other protective mechanism by PEG
may be due to its coupling with hydrophobic molecules
to produce non-ionic surfactants. Cheshmedjieva et al.
(1996) studied the eﬀect of addition of PEG 20 to egg
yolk based freezing medium on the cholesterol : phospholipid, sphingomyelin : phosphatidyl choline and
unsaturated : saturated fatty acids ratios in buﬀalo
spermatozoa. They concluded that PEG 20 added to
extender preserved the lipids of frozen buﬀalo spermatozoa. Further studies are required to ﬁnd out that PEG
20 may be a better option for the cryopreservation of
buﬀalo spermatozoa.
Sugars that are not capable of diﬀusing across a
plasma membrane, such as lactose, sucrose, raﬃnose,
trehalose or dextrans are also added to the extender as
non-permeable cryoprotectant. In these instances, the
sugars create an osmotic pressure, inducing cell dehydration and therefore, a lower incidence of intracellular
ice formation. These sugars also interact with the
phospholipids in the plasma membrane, reorganizing
the membrane which results in sperm that is better

suited to surviving the cryopreservation process (Molinia et al. 1994; Aisen et al. 2002). In early studies,
Ahmad and Chaudhry (1980) investigated the lactose or
fructose based extenders for cryopreservation of buﬀalo
semen. It was found that the diluent comprising 11%
lactose and 6% fructose achieved the best results as
tested by post-thaw motility and survivability. Ala Ud
et al. (1981) tested the post-thaw motility and survivability of buﬀalo spermatozoa frozen in homogenized
whole milk, Laiciphos (IMV), lactose or citrate-based
extender. They found that lactose-based extender gave a
better protection to sperm during the cryogenic procedure. Dhami and Sahni (1993) studied the eﬀect of 1%
raﬃnose in semen diluents (Tris–fructose–yolk–glycerol,
egg yolk–citrate–glycerol or lactose–egg yolk–glycerol)
on enzyme leakage (lactate dehydrogenase) from buﬀalo
spermatozoa during freezing. They found that the postthaw quality of spermatozoa was better with raﬃnose in
Tris-based extender compared to other extenders in
terms of release of lactate dehydrogenase.

Keeping in view the current international trends in
disease control, it is possible that extenders having
ingredients of animal origin (egg yolk) can be the source
of microbes ⁄ bacteria, consequently resulting in the
contamination of semen (Bousseau et al. 1998; MarcoJimenez et al. 2004; de Ruigh et al. 2006). In this regard,
LDL extracted from egg yolk (indirect use) or lecithin
from non-animal source like soya need to be tested as a
non-permeable cryoprotectant in extender for deepfreezing of buﬀalo spermatozoa.
Antibiotic
It is documented that bacteria in semen and their
control via addition of antibiotics in freezing diluents
may aﬀect the viability or fertility of cryopreserved
bovine spermatozoa (Thibier and Guerin 2000; Morrell

2006). Presence of bacteria in the ejaculates can aﬀect
fertilization directly (Morrell 2006), by adhering
to spermatozoa (Bolton et al. 1986; Wolﬀ et al. 1993;
Diemer et al. 1996), impairing their motility (Panangala
et al. 1981; Kaur et al. 1986) and inducing acrosome
reaction (El-Mulla et al. 1996). Microbes can also
have an indirect eﬀect by producing toxins (Morrell
2006).
Thus, in the use of AI, it is important to control
eﬃciently the population of microorganisms in the
semen. Conventionally, benzyl penicillin 1000 IU ⁄ ml
and streptomycin sulphate 1000 lg ⁄ ml alone or in
combination is commonly added to the freezing diluents
of buﬀalo bull semen (Sansone et al. 2000; Akhter et al.
2008). Regarding control of bacteriospermia in buﬀalo
bull semen with streptomycin and penicillin (SP), it was
found that it is not an eﬀective combination (Gangadhar
et al. 1986; Aleem et al. 1990; Hussain et al. 1990; Ali
et al. 1994; Amin et al. 1999). More recently, Ahmed
and Greesh (2001) and Ahmed et al. (2001a,b) found
that bacteria isolated from buﬀalo bull semen were
resistant to penicillin. Also SP was deleterious to postthaw quality of spermatozoa. They concluded that
gentamicin (500 lg ⁄ ml) or amikacin (500 lg ⁄ ml) or
norﬂoxacin (200 lg ⁄ ml) are the antibiotics of choice to
be added in extender for eﬃcient preservation of buﬀalo
spermatozoa.
Recently, Hasan et al. (2001) and Akhter et al. (2008)
investigated the eﬀects of a relatively new antibiotic
combination (gentamicin tylosin and linco-spectin,
GTLS) in extender on bacterial and spermatozoal

quality of preserved spermatozoa. They concluded that
GTLS is more capable than SP for bacterial control of
buﬀalo bull semen as judged by total aerobic bacterial
count and ⁄ or in vitro antibiotic sensitivity. Moreover,
GTLS is not detrimental to spermatozoal viability of
buﬀalo bull. It is relevant to mention that Andrabi et al.
(2001) have reported a better conception rate with
frozen–thawed semen having GTLS compared to SP
(55.2% vs 41.66%). It is therefore, suggested that GTLS
in extender is more eﬃcient for the preservation of
buﬀalo spermatozoa. Further, that testing of wider
range of new antibiotic is recommended in cryodiluents
for improvement in quality of frozen–thawed buﬀalo
spermatozoa.

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa

Other additives
Keeping in view the poor freezability of bubaline semen
attempts have been made to improve the basic buﬀers
developed to minimize the deleterious eﬀects of cryogenic
procedures. There are few scattered studies that have
used additives such as antioxidants, chelating agents,
metabolic stimulants, detergents etc. for improvement in
post-thaw quality of buﬀalo spermatozoa.
In this regard, Bhosrekar et al. (1990) studied the
eﬀect of addition of caﬀeine or triethanolamine lauryl

sulphate to Tris–citric acid-based extender. They reported that the addition of the detergent improved the
post-thaw spermatozoa motility. However, inclusion of
caﬀeine to extender did not made any improvement in
motility. It is believed that the protective eﬀect of
detergents may be exerted directly on the sperm membrane or is mediated through a change in the extending
medium such as emulsifying the egg yolk lipids to make
them more readily available to the plasmalemma during
cryopreservation (Graham et al. 1971; Arriola and
Foote 1987; Buhr and Pettitt 1996). On other hand,
the failure of caﬀeine to make any improvement is not
understood.
Dhami and Sahni (1993) studied the eﬀect of 0.1%
cysteine or 0.1% EDTA (sperm membrane stabilizer
and capacitation inhibitor) in semen diluents (Tris–
fructose–yolk–glycerol, egg yolk–citrate–glycerol or lactose–egg yolk–glycerol) on enzyme leakage (lactate
dehydrogenase) from buﬀalo spermatozoa during freezing. They found that the addition of cysteine or EDTA
to the experimental extenders did not improve the postthaw quality of spermatozoa in terms of release of
lactate dehydrogenase.
Singh et al. (1996) studied the eﬀect of addition of
ascorbic acid in the diluent on the quality of deep frozen
buﬀalo bull semen. They found that inclusion of
ascorbic acid (2.5 mM) in the semen diluent yielded a
signiﬁcantly higher post-thaw motility and survivability.
The antioxidant eﬀect of ascorbate is related to direct
vitamin E regeneration by reducing the tocopheroxyl
radical in the one-electron redox cycle (Packer et al.
1979; Dalvit et al. 1998). Later on, Kolev (1997) studied
the eﬀect of vitamin A, D and E in extender on motility,
survivability and acrosomal integrity of cryopreserved
buﬀalo bull spermatozoa. It was suggested that vitamin

E at 0.3 mg ⁄ ml exhibited the best eﬀects. It is wellknown that a-tocopherol inhibits lipid peroxidation
(LPO) in biological membranes, acting as a scavenger of
lipid peroxyl and alkoxyl radicals, thus preventing
oxidative damage in cryopreserved bovine semen
(Beconi et al. 1991).
Fabbrocini et al. (2000) suggested that for freezing
buﬀalo spermatozoa, addition of sodium pyruvate
(1.25 mM) to the extender resulted in signiﬁcantly better
post-thaw progressive motility and viability. The beneﬁcial eﬀect of pyruvate and a-ketoacids is attributed to
its antioxidant property.
Shukla and Misra (2005) studied diﬀerent antioxidants (a-tocopherol, ascorbic acid or n-propyl gallate)
added to Tris-based dilutor for improving freezability of
bubaline spermatozoa. They found that addition of
n-propyl gallate (15 lM) helped in retaining signiﬁcantly

561

high post-thaw motility and viability of spermatozoa. It
is noteworthy that propyl gallate is also an antioxidant.
It protects against oxidation by hydrogen peroxide and
oxygen-free radicals, in a catalytic manner by converting
hydrogen peroxide into water and oxygen.
Kumaresan et al. (2006) studied the eﬀects of addition
of oviductal proteins obtained from various stages of the
oestrous cycle to Tris-based extenders on spermatozoa
characteristics in buﬀaloes. They found that oviductal
proteins diﬀerentially aﬀected post-thaw sperm motility,
viability, acrosomal integrity, bovine cervical mucus
penetration test, hypo-osmotic sperm swelling test and
LPO level depending on the region of oviduct and the

stage of oestrous cycle at which the proteins were
obtained. Overall, it was implied that incorporation of
oviductal proteins in extender before freezing improved
functions and reduced the LPO levels in buﬀalo spermatozoa during cryopreservation. The beneﬁcial actions
conveyed by oviductal ﬂuid are presently unknown;
however, the identiﬁcation of catalase in cow oviductal
ﬂuid by Lapointe et al. (1998) suggests that it may be a
mechanism by which the oviductal ﬂuid reduces the
damage caused by reactive oxygen species to the
spermatozoa.
Recently, Shukla and Misra (2007) conducted a study
to improve buﬀalo semen cryopreservation with the
incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ⁄ ml) in
routinely used extender. They found that incorporation
of Bradykinin (2 ng ⁄ ml) in Tris-based extender might be
useful in improving the quality of frozen–thawed
bubaline spermatozoa as determined by live percentage,
motility and plasma membrane integrity. The exact
mechanism of action of Bradykinin is not yet fully
understood.
From the literature cited in this section, it appears
that there are some additives, which have some useful
eﬀects in terms of improvement in the quality of frozen–
thawed buﬀalo spermatozoa. It is relevant to mention
that most of these studies are preliminary. Therefore, it
is suggested that further research is required to establish
their beneﬁcial eﬀects on cryopreservation of buﬀalo
spermatozoa.
Semen processing
It is generally accepted that the cryopreservation process

itself reduces more than 50% of the sperm viability
(Watson 1979). During this process, the spermatozoa
are subjected to chemical ⁄ toxic, osmotic, thermal, and
mechanical stresses, which are conspicuous at dilution,
cooling, equilibration, or freezing and thawing stage.
The success of semen cryopreservation depends to a
notable degree on dilution rate. Originally, semen was
diluted to protect spermatozoa during cooling, freezing
and thawing, but the rate of dilution was often changed
for technical reasons, like to increase the number of
females, which could be inseminated with each ejaculate,
or to standardize the number of spermatozoa in each
dose of frozen–thawed semen (Salamon and Maxwell
2000). In farm animals, the semen has been diluted with
speciﬁc volumes of extenders or by diluting semen to a
speciﬁc spermatozoa concentration. Dilution rates of

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562

SMH Andrabi

1 : 1 to 1 : 12 have been successfully used for buﬀalo
semen. Perhaps a better of diluting semen, for comparison purposes, is based on the sperm concentration
(Purdy 2006). Reports of buﬀalo spermatozoa with
acceptable fertility, was with frozen samples ranging
from 120 · 106 to 30 · 106 cells ⁄ ml (Tahir et al. 1981;
Andrabi et al. 2006).

After dilution, the semen is cooled to a temperature
close to 4°C or 5°C. Cooling is a period of adaptation of
spermatozoa to reduced metabolism. Extended semen is
cooled slowly to avoid potential of cold shock. Cold
shock is believed to impair function of membrane
proteins that are necessary for structural integrity or
ion metabolism (Watson 2000). Major changes in bovine
spermatozoa during this phase occur near 15 to 5°C, and
do not happen below 0°C (Watson 2000). Rapid cooling
reduces the rate of fructose breakdown, oxygen uptake,
and ATP synthesis by the sperm, which results in the
loss of energy supply and motility (Blackshaw and
Salisbury 1957; Wales and White 1959). Furthermore,
cold shock may increase calcium uptake by sperm
(White, 1993). However, some think that a faster cool
will not create problem if the semen is extended in an
ideal buﬀering system (Marshall 1984). It has been
empirically determined that cooling cattle bull spermatozoa from body temperature to 5°C performed at a rate
of 10°C ⁄ h has minimum deleterious eﬀects (Parks 1997).
In this regard, Dhami et al. (1992) studied the eﬀect of
cooling rates (5, 30, 60 and 120 min from 10 to 5°C vs
120 min from 28 to 5°C) on the deep freezing of buﬀalo
semen diluted in Tris-based extender. Their results
suggest that buﬀalo semen can be frozen successfully
after 30 min of cooling at 10°C as judged by motility
and survivability.
Regarding equilibration, it is traditionally taken as
the total time during which, spermatozoa remain in
contact with glycerol before freezing. At this stage,
glycerol penetrates into the sperm cell to establish a

balanced intracellular and extracellular concentration. It
should not be overlooked that the equilibration includes
the concentration balance not only of glycerol, but also
of the other osmotically active extender components
(Salamon and Maxwell 2000). Therefore, this phenomenon interacts with the type of extender (buﬀer and
cryoprotectant) used and could easily interact with other
cryogenic procedures (Marshall 1984). In this regard,
Tuli et al. (1981b) examined equilibration of buﬀalo
semen diluted in Tris or citric acid-based extender for 2,
4 or 6 h. They found that post-thaw sperm survivability
was better after 4 h equilibration than after 2 or 6 h.
Talevi et al. (1994) cooled buﬀalo semen from 28°C to
5°C in 15 min and then equilibrated at 5°C for 1 h
45 min (fast cooling) or cooled it in 1 h and equilibrated
for 1 h (slow cooling). They found that post-thaw sperm
motility was signiﬁcantly higher using the slow than the
rapid cooling method. Conversely, the rate of cooling
had no signiﬁcant eﬀect on acrosome integrity. Dhami
et al. (1996) conducted a study to determine the relative
eﬃcacy of four cooling rates (10 ⁄ 30°C to 5°C; 1 and 2 h
each) and two equilibration periods at 5°C (0 and 2 h)
for cryopreservation of buﬀalo ejaculates. They concluded that slow cooling of straws from 30 to 5°C for
2 h compared with faster cooling (1 h) or lower initial

temperature (10°C) and 2 h of equilibration at 5°C
appeared necessary for successful cryopreservation of
buﬀalo semen as determined by survivability and
fertility.
Of considerable importance for the cryopreservation
is the cooling ⁄ freezing rate in the critical temperature

range ()5 to )50°C) that determines whether the
spermatozoa will remain in equilibrium with their
extracellular environment or become progressively supercooled with the increasing possibility of intracellular
ice formation (Kumar et al. 2003). During slow cooling,
the dehydration of the spermatozoa can proceed to the
point of osmotic equilibrium between intracellular and
extracellular space i.e., cellular dehydration will be
maximal. While raising the cooling rate too much, the
dehydration is not fast enough to prevent occurrence of
intracellular ice nucleation. However, if the cooling rate
is within the required values (50–100°C) this results in
less excessive intracellular dehydration, less excessive
intracellular solute concentrations and less shrinkage of
the cells (Mazur 1984; Woelders 1997). Moreover, at
optimum cooling ⁄ freezing rates, the spermatozoa remain vulnerable to the unfavourable conditions for a
shorter period of time (Woelders 1997). It is worth
mentioning that for cattle bull semen currently a
freezing rate of ‡40°C is practiced in general for
cryopreservation during the critical temperature zone
(Anzar et al. 2002).
Sukhato et al. (2001) determined the eﬀects of freezing rate and intermediate plunge temperature (cooling at
10, 20 or 30°C ⁄ min each to )40, )80 or )120°C before
being plunged into liquid nitrogen) on post-thaw quality
and fertility of buﬀalo spermatozoa. They found that
cooling ⁄ freezing spermatozoa from 4 to )120°C, either
at 20 or 30°C ⁄ min yielded better progressive motility
and fertility rate. Bhosrekar et al. (1994) compared the
conventional (over liquid nitrogen in static vapour for
10 min) and control (programmable) freezing methods
for buﬀalo bull semen. It was concluded that freezing at

the rate of 17.32°C ⁄ min between +4°C and )40°C with
programmable freezer produced better quality frozen
semen than the conventional method of freezing. More
recently, Rasul (2000) examined the eﬀect of freezing
rates on post-thaw viability of buﬀalo spermatozoa
extended in Tris–citric acid-based extender. The freezing
rates examined between 4 and )15°C were 3 or
10°C ⁄ min, whereas the freezing rates investigated
between )15 and )80°C were 10, 20 or 30°C ⁄ min. It
was concluded that the diﬀerent freezing rates tested
gave similar results in terms of post-thaw spermatozoal
viability as judged by visual and computerized motilities, motion characteristics, plasma membrane integrity
and intactness of acrosomal ridge.
In the freeze–thaw procedure, the warming phase is
just as important to the survival of spermatozoa as the
freezing phase. Spermatozoa that have survived cooling
to )196°C still face the challenge of warming and
thawing, and thus must traverse twice the critical
temperature zone i.e., from )5 to )50°C (Marshall
1984). The thawing eﬀect depends on whether the rate of
cooling has been suﬃciently high to induce intracellular
freezing, or low enough to produce cell dehydration. In
the former case, fast thawing is required to prevent

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Factors Aﬀecting Cryopreservation of Buﬀalo Spermatozoa

recrystallization of any intracellular ice present in the

spermatozoa. Spermatozoa thawed at a fast rate may
also be exposed for a shorter time to the concentrated
solute and cryoprotectant-glycerol, and the restoration
of the intracellular and extracellular equilibrium is more
rapid than for slow thawing (Salamon and Maxwell
2000). Also leaving straws in high temperatures for too
long time may result in pH ﬂuctuation and subsequently
protein denaturation and cell death. A practical thaw
for cattle bull spermatozoa, recommended by most AI
organizations, is as 35°C water bath for at least 30 s
(Marshall 1984). For cryopreservation of buﬀalo spermatozoa in Tris-based extender, Rao et al. (1986) tested
two thawing rates (37°C for 30 s and 75°C for 9 s). They
concluded that the best value for post-thaw motility was
observed for semen thawed at 37° for 30 s. Dhami et al.
(1992) studied the eﬀect of thawing rates (40°C for 60 s,
60°C for 15 s and 80°C for 5 s) on post-thaw motility of
buﬀalo spermatozoa cryopreserved in Tris-based
extender. They reported that thawing at 60°C for 15 s
yielded a higher sperm motility compared to other rates.
In another study, Dhami et al. (1996) determined the
thawing rates for buﬀalo semen. The thawing rates
investigated were 4°C for 5 min, 40°C for 1 min or 60°C
for 15 s. They concluded that thawing of semen at 60°C
for 15 s yielded high post-thawing spermatozoal recovery and longevity. Sukhato et al. (2001) determined the
eﬀect of thawing rates on motility and acrosome
integrity of buﬀalo spermatozoa. Thawing of spermatozoa was performed at the rate of (rapid) 1000°C or
(slow) 200°C ⁄ min. They concluded that rapid thawing
was superior to slow warming.
The above-mentioned studies demonstrate that an
eﬀective cryopreservation procedure for buﬀalo spermatozoa can be derived by the systematic examination

of various cryobiological factors. Therefore, from these
studies the cryogenic procedures for buﬀalo semen can
be outlined as; cooling from 37 or 39 to 4°C at the rate
of 0.2–0.4°C ⁄ min, equilibration, at least 2 h at 4°C,
freezing of straws approximately 4 cm above liquid
nitrogen for 10–20 min, or by the fast freezing rates
(programmable freezing), and thawing at 45–60°C for at
least 15 s. However, there is still need to improve the
processing techniques for cryopreservation of buﬀalo
spermatozoa. It is suggested that to devise eﬃcient
cooling ⁄ freezing rates for buﬀalo spermatozoa, studies
involving use of a cryomicroscope should be carried out,
as this will permit a direct and continuous viewing of
spermatozoa, while the temperature is controlled accurately (Medranol et al. 2002).
Season of semen collection
Freezability of buﬀalo semen can also be aﬀected by the
season of collection i.e., by environmental factors like
temperature, humidity and day length in a particular
season. For the ﬁrst time Tuli and Mehar (1983) studied
the seasonal variation in freezability of buﬀalo semen
diluted in Tris-based extender. They found that post-thaw
spermatozoa motility, signiﬁcantly increased in winter
than summer season. After that, Heuer et al. (1987)
studied the eﬀect of season on in vivo fertility of frozen
buﬀalo semen diluted in chemically deﬁned buﬀers. They

563

reported that semen collected in November (winter)
produced signiﬁcantly higher conception rate than semen

collected in June (summer) over a total of 3220 inseminations in both seasons (40.9 vs 34.0%). They attributed
40% of the observed seasonality of buﬀalo fertility to the
male. Bhavsar et al. (1989a) also studied the monthly
variations in freezability and fertility of buﬀalo bull
semen. They found that fertility of semen collected, frozen
and inseminated during season from July to January
(monsoon or late wet summer to autumn and winter) was
signiﬁcantly higher than ejaculates processed and inseminated during the season from February to June (spring to
early dry summer).
Sagdeo et al. (1991) studied the seasonal variations
in freezability of buﬀalo bull semen. They found from
the data of over a 4-year period that the season
signiﬁcantly aﬀected the post-thaw sperm motility, and
values being highest in ejaculates frozen in the winter
and lowest in summer. Similarly, Bahga and Khokar
(1991) studied the seasonal variations in freezability of
buﬀalo bull semen. They found that post-thaw semen
motility was signiﬁcantly aﬀected by season of collection, being lowest in summer and highest in winter
(December–January). Younis et al. (1998) studied the
freezability of semen collected during the low breeding
season (May–July) and the peak breeding season
(September–November) in young (3–4 years), adult
(6–8 years) and old (12–15 years) buﬀalo bulls. They
reported that post-thaw motility and liveability of
spermatozoa frozen in Tris-based extender were significantly higher in adult bulls during the peak breeding
season. In addition, the sperm abnormalities and
deleterious enzymatic activity in frozen–thawed semen
were signiﬁcantly higher during the low breeding
season than in the peak breeding season.
Recently, Koonjaenak et al. (2007a) studied the

seasonal eﬀect on quality of frozen–thawed buﬀalo
spermatozoa diluted in Tris-based buﬀer. They compared post-thaw sperm quality over three seasons of the
year (rainy: July–October; winter: November–February;
and summer: March–June), with distinct ambient temperature and humidity. Their conclusion was that postthaw plasma membrane integrity and stability were
signiﬁcantly better in ejaculates processed during winter
than in samples processed during the other seasons of
the year. From the above-mentioned studies, it is evident
that there is a higher loss of viability and fertility during
the process of cryopreservation in summer, thus conﬁrming that vitality of buﬀalo spermatozoa remain
comparatively poorer during this season. Moreover, it is
suggested that to increase fertility rate in buﬀalo, semen
should be collected and preserved during cooler months
and used for AI all over the year.
In another study, Koonjaenak et al. (2007b) investigated the frozen–thawed buﬀalo sperm nuclear DNA
fragmentation by ﬂowcytometry and head morphology
over three seasons in tropical Thailand (the rainy season,
July–October; winter, November–February; and summer, March–June). They found that the DNA fragmentation index (DFI) values varied statistically among
seasons, being lower in the rainy season than in winter or
summer, and were aﬀected by the year of semen collection
and processing. The proportion of morphologically

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564

SMH Andrabi

abnormal sperm head shapes was low, with no signiﬁcant
diﬀerences between seasons. However, DFI was signiﬁcantly related to the proportion of loose abnormal sperm

heads. It was concluded that frozen–thawed buﬀalo
sperm chromatin is not critically damaged by cryopreservation or aﬀected by the seasonal variations in temperature and humidity seen in tropical Thailand.
There is possibility that a seasonal variation in the
biochemical composition of seminal plasma and ⁄ or
spermatozoa may occur as it does in other farm animals
(Cabrera et al. 2005; Argov et al. 2007; Koonjaenak
et al. 2007a). Recently, Argov et al. (2007) have
reported that cattle semen samples collected during the
summer and considered to be of good quality had
alterations in lipid concentration, fatty-acid composition and cholesterol level. In addition, they provided the
ﬁrst evidence for the existence of a very-low-density
lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular
lipids. Interestingly, the expression of VLDLr was
threefold greater in samples collected during the winter
than in those collected in the summer. Therefore, it is
suggested that such modiﬁcations may explain, in part,
the reduced freezability of buﬀalo semen collected
during the summer.
Few scattered reports are available that describe the
diﬀerences in chemical composition of buﬀalo seminal
plasma and spermatozoa under diﬀerent climatic
conditions (Singh et al. 1969; Mohan et al. 1979;
Sidhu and Guraya 1979). However, the information
given in these studies are insuﬃcient to explain the
variation in freezability of buﬀalo spermatozoa during
the diﬀerent seasons. Therefore, detailed studies should
be carried out to ascertain the biochemical or structural diﬀerences in seminal plasma, spermatozoa, or
plasmalemma, which might be inﬂuencing the freezability of buﬀalo spermatozoa during the diﬀerent
months ⁄ seasons.

Conclusions
Viability and fertility of frozen–thawed buﬀalo bull
spermatozoa is considerably lower than that of cattle
bull. Several buﬀers, cryoprotectants, antibiotics, other
agents and various cooling, freezing and thawing rates
initially developed for cattle bull spermatozoa have been
used, at times with contrasting results. Therefore, a
better understanding of the fundamental principle of
cryopreservation of buﬀalo spermatozoa is necessary
according to the speciﬁc requirements. Moreover, there
is a need to develop biochemically deﬁned extenders and
cryogenic procedures that may result in improvement in
viability and fertility of frozen–thawed buﬀalo spermatozoa. Besides this, the season during which the semen is
collected should also be considered as a variable
aﬀecting quality of cryopreserved buﬀalo spermatozoa.

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Submitted: 04 Jun 2008
Author’s address (for correspondence): Dr SMH Andrabi, Animal
Reproduction Laboratory, Animal Sciences Institute, National
Agricultural Research Centre, Islamabad 45500, Pakistan. E-mail:

Ó 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag

Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa

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