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Storage of buffalo Bubalus bubalis semen
G. Sansone
), M.J.F. Nastri, A. Fabbrocini
Dept. of Fisiologia Generale ed Ambientale, UniÕersita degli Studi di Napoli Federico II,`
Via Mezzocannone 8, 80134 Naples, Italy
Abstract
Characteristics of buffalo semen, diluents used for liquid storage, aspects involved in freezing Ž . and thawing of semen are reviewed, and fertility results after artificial insemination AI with frozen–thawed semen are given.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Buffalo; Semen; Spermatozoa; Diluent; Freezing; Thawing
1. Introduction
Ž
The world’s buffalo population is estimated to be more than 140 million Vale,
.
1997 . But despite its importance for the production of milk, meat and leather, it has not received sufficient attention regarding the improvement of breeding practices. Recent studies have shown that buffalo semen can be preserved just like bovine semen. There are a number of diluents and cryoprotectants, which are suitable for freezing buffalo semen, but there is limited information available on the semen characteristics of the species.
The success of semen storage depends on numerous factors which may be peculiar to each species and are optimised according to the type of semen to be preserved. Buffalo spermatozoa are more susceptible to hazards during freezing than cattle spermatozoa
ŽRaizada et al., 1990 . These hazards can be minimised by optimising the cooling and.
freezing rates and using appropriate diluting media in which spermatozoa are frozen
ŽKumar et al., 1992a . This review deals with the characteristics, methods of processing.
and storage of buffalo semen.
)Corresponding author. Tel.:q39-81-552-7736; fax:q39-81-542-4848.
Ž .
E-mail address: giosanso@cds.unina.it G. Sansone .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
2. Collection and characteristics of semen
Semen samples are collected from trained buffalo bulls by using an artificial vagina maintained at temperature between 398C and 418C. The male buffalo is perhaps the easiest domestic species to be trained to serve an artificial vagina. The semen is usually collected early in the morning, before feeding, and each collection consists of two ejaculates taken within a minimum interval of 30 min.
The characteristics of semen generally examined are: colour, volume, concentration, motility, viability, acrosomal and membrane integrity, and percentage of abnormal
Ž .
spermatozoa. Den Daas 1992 reported that the relationship between the semen characteristics and fertility has often been inconsistent. Characteristics of semen are shown in Table 1.
2.1. Colour andÕiscosity
The colour and viscosity of semen depend on variations in concentration of spermato-zoa. Buffalo semen generally varies from a milky white to creamy colour, with a slight
Ž . Ž .
tinge of blue Vale, 1994a . Kumar et al. 1993a found significant differences in viscosity of ‘‘static’’ and ‘‘motile’’ ejaculates, but not between different bulls.
2.2. Volume
The volume of the buffalo semen is measured immediately after collection. It varies, depending on breed and age of the bull. Young bulls give 1–3 ml volumes of semen
Ž .
while older bulls give 6 ml Vale, 1994a . No differences have been found among
Ž .
buffaloes bred in different countries Alexiev et al., 1994; Misra et al., 1994 .
2.3. pH of semen
Ž
Buffalo semen has a pH within the range of 6.4–7.0 Rattan, 1990; Kumar et al.,
.
1993b; Aguiar et al., 1994; Vale, 1997 .
Table 1
Ž .
Characteristics of buffalo semen collected by artificial vagina according to Vale, 1994a Characteristics
Colour white, milky white, with light blue tinge
Ž . Ž .
Concentration cellsrml 600=10 to 1200=10
Ž .
Live sperm % )70
Ž .
Abnormal sperm % -70
2.4. Concentration of spermatozoa
Most routine measurements of concentration are made by a spectrophotometer or
Ž .
haemocytometer. Woelders 1991 suggests that the fluorimetric measurement of the amount of DNA might give a reliable estimate of concentration of spermatozoa.
Ž . 6
Buffalo bulls bred in Bahia Brazil showed a concentration of 1166.3"17.5=10
Ž . Ž .
cellsrml Aguiar et al., 1994 . Galli et al. 1993 observed concentrations from 690.6"187.9=106 to 1290.7"100=106cellsrml in water buffaloes bred in Italy. In
Ž .
Murrah buffalo bulls bred in India, Kumar et al. 1993a found sperm concentrations from 524.1"20.7=106 to 1031.4"28.7=106 cellsrml. Similar results were
ob-Ž .
served in Murrah buffalo by Rattan 1990 .
2.5. Motility of spermatozoa
Motility is routinely assessed by visual estimate of the percentage of motile cells. A small drop of semen is placed on a dry slide maintained at 378C, and examined at a magnification of 40- or 100-fold. The Makler chamber is equipped with a grid which facilitates cell counting.
Ž .
In the last 10 years, the introduction of computer-aided semen analysis CASA has enabled those working in the field to use new parameters in assessing sperm motility. In a semen sample, there can be variations in the degree of progressive movement of cells and in lateral dislocation of sperm heads. The CASA system can evaluate parameters like speed, direction and the beat cross frequency of sperm cells. Forward moving spermatozoa and only those which traced five straight tracks and had a minimum
Ž
velocity of 50 mmrs have been considered Del Sorbo et al., 1992; Fabbrocini et al.,
. Ž .
1995 . Aguiar et al. 1994 observed 78.6"5.6% motile spermatozoa in semen of buffalo bulls bred in the Brazilian states of Minas Gerais and Bahia. Water buffaloes in
Ž .
Italy showed a variation in motility from 40"2% to 82"5% Galli et al., 1993 .
Ž .
Kumar et al. 1993a found that in semen of Murrah buffalo bulls bred in India, the percentage of motile spermatozoa varied from 60.8"1.5% to 69"4%, while the occurrence of non-motile samples was about 30%. However, non-motile spermatozoa regained their motility after dilution.
2.6. Viability
The percentage of live spermatozoa determines the quality of the ejaculate. Semen with more than 30% initial dead spermatozoa may not be suitable for storage and freezing. Differential staining techniques have been used for determination of live and
Ž .
dead spermatozoa Rochwerger and Cuaniscu, 1992 .
2.7. Abnormal spermatozoa
Abnormal spermatozoa are detected by staining methods and are usually classified as
Ž .
Ž .
buffaloes, most abnormalities were found on sperm heads 5.78"2.1% , while middle-piece abnormalities were less than 1% and abnormal tails varied from 3.92"1.0% to
Ž .
5.7"0.4%. Occurrence of cytoplasmic droplet was less than 1% Saeed et al., 1990 . Similar proportions of abnormalities were observed in semen of Brazilian buffaloes
ŽAguiar et al., 1994 and Murrah buffaloes Kumar et al., 1993a . The latter authors. Ž .
suggested that semen showing over 15–20% abnormal spermatozoa should be examined for their fertility.
2.8. Acrosomal and membrane integrity
Most workers examined the acrosome abnormalities by using the Giemsa stain
Ž
technique Bhosrekar et al., 1994; Raizada et al., 1990; Ramakrishnan and Ariff, 1994;
. Ž
Rao et al., 1990 or fluoresceinated lectins Bawa et al., 1993; Chachur et al., 1997;
.
Cross and Meizel, 1989; De Leeuw et al., 1991 .
More than 90% of spermatozoa were observed with intact acrosome in semen of
Ž .
buffalo bulls bred in Bahia Aguiar et al., 1994 and Murrah buffalo bulls after Giemsa
Ž . Ž .
staining Kumar et al., 1993a . Talevi et al. 1994 reported similar results for water
Ž .
buffalo using a fluoresceinated lectin. Fabbrocini et al. 1996 used fluoresceinated
Ž .
lectin, FITC-labeled Maclura pomifera Agglutinin MPA , that binds to lectin-similar receptors on the cell surface to detect changes in the surface glycoconjugates. Three
Ž . Ž .
different sub-populations were found: 1 cells with a coloured acrosome and tail, 2
Ž .
cells with the external border of the acrosome and the tail coloured, 3 uncoloured cells
Ž . Ž . Ž .
Table 2
Ž . Ž .
Composition of buffalo semen according to Vale 1997 mgr100 ml
Carbohydrates Fructose 623 Siliac acid 133
Nitrogenous costituents Total nitrogen 684 Non-protein nitrogen 136
Organic acids Citric acid 441 Ascorbic acid 6.2
Inorganic costituents Total P 108, Inorganic P 5.4 Ca 44.4, Mg 4.3
Žsee Fig. 1 . In semen of good quality, cells presenting pattern 1 were most common,.
while patterns 2 and 3 occurred in less than 20% of spermatozoa.
2.9. Biochemical characteristics of semen
The biochemical constituents and the activity of specific enzymes in buffalo semen are shown in Tables 2 and 3. Zinc is closely linked to sperm morphology, physiology and biochemistry. The biochemical functions in which Zn has been implicated include enzymatic function, the protein and carbohydrate metabolism and possibly sperm motility. Also, Zn concentration both in spermatozoa and seminal plasma is one of the regulating mechanisms of cations between the intra and extra cellular compartment, and
Ž .
hence it may affect the sperm metabolism and motility Ahmed and El Tohamy, 1997 . The latter workers determined concentrations of Zn in both spermatozoa and seminal plasma and correlated the concentrations with the age of buffalo bulls and semen quality. No significant changes were found in the amount of zinc in spermatozoa, but there was a marked decrease in seminal plasma with the advancement of age. The Zn concentration in seminal plasma averaged 86.88 mmolrl, whereas its concentration in sperm cells was greater and averaged 255.55mmolrg, or 14.3mmolrcell. The increase in motility and the decrease in percentage of abnormalities are correlated to the increase
Table 3
Ž
Physical and biochemical characteristics of whole semen and seminal plasma modified by Ibrahim et al.,
.
1985
Characteristics Whole semen Seminal plasma
Ž .
Osmolarity mosMrkg 293.33"3.39 283.75"2.31
Ž .
Total proteins gr100 ml 3.10"0.10 2.86"0.14
Ž .
Total lipids mgr100 ml 321.15"18.41 260.86"12.52
Ž .
Fructose mgr100 ml 547.08"61.24 684.60"81.14
Ž .
Citric acid mgr100 ml 368.73"14.82 466.33"31.66
Ž .
Sodium mgr100 ml 260.63"8.81 258.58"13.65
Ž .
Potassium mgr100 ml 153.50"2.68 154.83"3.27
Ž .
Calcium mgr100 ml 32.04"2.77 32.42"3.10
Ž .
Magnesium mgr100 ml 6.17"0.41 6.46"0.39
Ž .
Chloride mgr100 ml 196.57"2.45 224.06"2.60
Ž .
Inorganic phosphatase mgr100 ml 17.02"1.67 12.75"1.09
Ž .
Acid phosphatase Ur100 ml 225.00"2.99 230.46"1.48
Alkaline phosphatase 326.05"2.16 331.20"2.60
Ž .
in Zn concentration in the spermatozoa, while no relation was found between Zn concentration in the seminal plasma and motility of spermatozoa.
2.10. Coating antigens
The sperm coating antigen pattern on fresh buffalo spermatozoa was used as a
Ž .
parameter to evaluate the quality of the semen by Bergamo et al. 1991 .
2.11. Semen protein pattern
Seminal plasma contains several components that are determined by the contribution of the accessory sexual glands. The electrophoretic pattern of seminal plasma on
Ž .
polyacrylammide gel SDS-PAGE may be used to identify the normal physiology of the reproductive glands, and thereby to select the donor bulls whose semen could be used
Ž .
for artificial insemination AI .
( )
2.12. Alkaline phosphatase KPH
It has been claimed to have a possible role of sperm coating antigen with a masking
Ž
function of the receptors, whose activity, however, has not yet been defined Odierna et
.
al., 1990 .
2.13. Sperm–oocyte interaction
Assessments of spermatozoa are mainly based on examination of their motility, concentration and morphology. However, these characteristics do not give reliable indication on their fertilising capability. Therefore, a method based on spermatozoon–egg
Ž . Ž .
interaction has been proposed Gamzu et al., 1994 . Di Matteo 1997 developed a simple technique to assay the capacity of buffalo spermatozoa to bind to the zona pellucida. As buffalo oocytes are difficult to obtain, due to the fact that females of this species are slaughtered only in old age or in illness, bovine oocytes either preserved in
Ž .
saline solution ‘‘salt-stored’’ oocytes or matured in vitro were used. The results showed that for a rapid evaluation of fresh or frozen–thawed buffalo spermatozoa ‘‘salt-stored’’ bovine oocytes can be used, which gave similar results to buffalo oocytes. Zona-free hamster oocytes may be more convenient to obtain and to use for
Ž .
functional tests Ramesha et al., 1993 . The zona-free hamster oocytes permit entry of
Ž .
spermatozoa of many mammals including buffalo provided the spermatozoa have completed capacitation and acrosomal reaction, and can therefore be used for assessing
Ž .
the fertilising capacity of spermatozoa Barnabe et al., 1997 .
3. Factors influencing the quality of semen
Photoperiod is an important environmental factor influencing reproduction and sexual
Ž .
effect not only on the libido, but also on the quality and freezability of buffalo semen. These effects need to be studied in all areas where buffaloes are bred, due to the considerable environmental differences.
In the temperate regions of the world, it has been found that the semen is of better
Ž
quality during the winter and spring than in summer and autumn Galli et al., 1993;
.
Mohan and Sahni, 1990 . In the tropical regions, the quality of semen was observed to be satisfactory during the rainy season. In the warm and humid tropical Amazon region,
Ž .
the best time to obtain semen is between January and June Vale, 1994b . Buffaloes are very sensitive to heat stress, thus a decline in the quality of semen is a common finding during the hot season of the year.
The management of the buffalo bulls, including feeding and housing, also has an
Ž .
influence on the production and quality of the semen Chinnaiya and Ganguli, 1990 . Another influencing factor is the age of the buffalo bull. Semen of best quality, with
Ž
regard to sperm morphology, was observed in 3- to 5-year-old Murrah Kumar et al.,
. Ž .
1993a; Chinnaiya and Ganguli, 1990 and Nili–Ravi buffalo bulls Saeed et al., 1990 .
Ž
There were significant differences in the quality of semen of individual bulls Mohan
.
and Sahni, 1990; Galli et al., 1993; Kumar et al., 1993a .
4. Liquid storage of semen
4.1. Diluents examined
Buffalo semen can be stored at 58C for up to 72 h without significant decrease in motility, if it is diluted with media which have the same composition as those used for
Ž .
freezing Dhami et al., 1994 .
Cow milk is a widely used diluent for liquid storage of buffalo semen. Kumar et al.
Ž1993b recommended that before use for dilution, the milk should be heated, cooled.
overnight in a refrigerator, then the fat layer removed and the milk reheated in a water bath for 10–12 min. After repeated cooling, the remaining fat should be removed by filtration through cotton wool.
wŽ . x
Tris hydroxymethyl aminomethane , egg yolk–citrate and egg yolk–lactose are also popular diluting media for storage of buffalo semen. After storage of semen in milk, tris-and citrate-based diluents at 58C for 24 h, the decrease in motility was similar in the
Ž
three media, but after 48 h only milk and tris were able to maintain motility Kumar et
. Ž .
al., 1992a . Dhami et al. 1994 examined the relative efficacy of tris-, citrate- and lactose-based diluents, and found that the best survival after 72 h at 58C was in tris buffer. The proportion of motile spermatozoa improved in all three diluents after
Ž .
addition of 0.1%L-cysteine or ethylenediaminetetra-acetic acid, tetrasodium salt EDTA .
Ž .
Examination of different glycerol concentrations 3%, 6%, 9% in milk-, tris- and
Ž .
citrate-based extenders Kumar et al., 1992a revealed that for 24 h storage at 58C,
Ž
glycerol was not required in the extender; but in case of a longer period of storage 72 h
.
Ž .
In studies of different egg yolk 1%, 2.5%, 5%, 10%, 20% concentrations in
Ž .
tris-based diluent, the low levels 1% and 2.5% showed the best sperm survival rates
Ž .
during 72 h storage at 58C Sahni and Mohan, 1990 . There was no improvement in viability of spermatozoa after 72 h stored at 58C when 5%, 10% and 20% egg yolk was included in milk- or tris-based extenders, but in the latter diluent the spermatozoa
Ž .
survived better in the presence of 10% or 20% than 5% egg yolk Kumar et al., 1993b . Published reliable reports on fertility of liquid stored buffalo semen seem not to be
Ž .
available editorial comment .
5. Frozen storage of semen
5.1. Diluents
An important factor influencing frozen storage of semen is the composition of the medium used for dilution of semen before freezing. The diluent is usually composed of a buffer medium to which cryoprotectants and other substances are added, which protect the spermatozoa during freeze–thawing. Antibiotics are commonly added to the exten-der.
Ž .
In studies of Dhami et al. 1994 , tris–fructose–egg yolk, citrate–egg yolk and lactose–egg yolk diluents did not differ in their ability to protect spermatozoa against freeze–thawing damages. However, the tris-based diluent yielded higher post-thaw
Ž .
Ž .
motility compared with the other two media 54.1%, 46.5% and 41.6%, respectively .
Ž .
Other workers Raizada et al., 1990; Chinnaiya and Ganguli, 1990 also found no difference in post-thaw motility of spermatozoa frozen in tris- or citrate-based extenders. Both tris- and lactose-based media were superior to the milk diluent as determined by
Ž .
post-thaw motility Akhtar et al., 1990 .
In other in vitro comparisons of diluents, based on post-thaw motility of spermatozoa,
Ž .
milk was better than tris Galli et al., 1993 , or both these media performed better than
Ž .
the citrate-based extender Kumar et al., 1992a . However, in a subsequent test, the performance of citrate medium was similar to that of commercial milk-based diluent,
Ž
and was also found sufficiently clear for computerised analysis of motility see Figs. 2
. Ž .
and 3 Del Sorbo et al., 1995a .
wŽ . .
Tes Hydroxymethyl aminoethan is another buffer used for freezing buffalo semen,
Ž
and proved to be of equal value to tris- and glycine-based diluents Oba et al., 1994;
.
Chachur et al., 1997 .
Ž .
Galli et al. 1993 suggested that the differences regarding in vitro performance of the various extenders, which may not emerge in tests with bovine semen, indicate that buffalo spermatozoa are more prone to freezing stress. The poor freezability may be correlated to the low membrane phospholipid content and to its loss during freeze–thaw-ing.
5.1.1. Cryoprotectants
Both egg yolk and glycerol are used in combination as cryoprotectants. The lecithin and lipoprotein contents in egg yolk contribute to the preservation of the lipoprotein
Ž .
sheath of the sperm cell Kumar et al., 1992a . However, in addition to its protective
Ž .
action against cold shock, the egg yolk also stimulates the enzyme system of spermato-zoa. This results in deamination of certain specific amino acids normally present in the dialysable fraction of egg yolk and yields hydrogen peroxide, which is toxic to
Ž .
spermatozoa during storage under aerobic conditions Sahni and Mohan, 1990 . For this reason, the egg yolk may be dialysed before addition to the extender. Little attention has been paid to the level of egg yolk necessary for freezing buffalo semen, and the majority of workers are using concentrations around 20%. It should be noted, however, that as the yolk concentration is increased in the diluent, the pH of the medium decreases and tends towards the acidic side. This may also be the reason for the depressing effect of higher amounts of yolk on motility of thawed spermatozoa. The toxic effect of egg yolk may be
Ž .
combined with toxicity of dead spermatozoa Shannon, 1972 .
Ž .
Sahni and Mohan 1990 examined various levels of egg yolk in a tris–glycerol-based freezing diluent and found that egg yolk beyond 5% did not show any significant improvement in post-thaw motility. This indicated the scope of reducing the yolk level from 20% to 5% without adversely affecting the freezability of buffalo semen. In the
Ž .
absence of egg yolk, none of the diluents used tris-, milk- and citrate-based gave protection to spermatozoa during freeze–thawing. After addition of egg yolk, post-thaw motility improved significantly, but there was no further increase for concentrations
Ž .
higher than 5% in the three buffers Kumar et al., 1993b .
Glycerol is generally added to the extender for freezing buffalo semen in concentra-tions from 6% to 7%, but attempts have been made to reduce the amount, or substitute it with other cryoprotective substances. Reduction of glycerol to 3% or 2%, and concentra-tions above 7% decreased the post-thaw motility of spermatozoa in the diluents tested
ŽKumar et al., 1992a; Ramakrishnan and Ariff, 1994; Nastri et al., 1994 ..
The cryoprotective effects of six sugars — glucose, xylose, raffinose, fructose,
Ž .
sucrose and cheeni a high molecular weight sugar containing 99.5% sucrose each at
Ž .
1%, 1.5% and 2% concentrations, were examined by Kumar et al. 1992b . They found that the protective effect of sugars depended on the type of diluent used for freezing. In milk diluent, fructose and sucrose gave the best protection, as judged by post-thaw motility results, due probably to the synergy action of lactose present in milk. Some protection to spermatozoa was given by the sugars at 2% concentration in tris diluent, and none in citrate-based medium. The high molecular weight sugars may play a cryoprotective role by altering the permeability of the cell membrane and by maintaining the electrolyte balance. However, the best post-thaw motilities were never higher than 30% when sugar was used as the sole cryoprotectant.
The use of extenders of vegetable origin for the preservation of live cells has long been known. Some vegetable derivatives, like coconut water and tomato juice, seem to have properties that protect bovine and caprine spermatozoa. Coconut water has been shown to be an alternative for storage of bovine semen, as the phytohormones it contains
Ž
protect the spermatozoa against the phospholipase A enzyme in the ejaculate Nunes,
. Ž . Ž .
1993 . Nunes et al. 1996 reported that indole-3-acetic acid IAA in coconut water appeared to be the main protective substance for spermatozoa. The suitability of coconut
Ž .
5.1.2. AdditiÕes to freezing diluents
Many attempts have been made to improve the basic buffers developed in the early 1950s by inclusion of additives such as vitamins, amino acids, chelating agents, enzymes, metabolic stimulants and others.
Seminal plasma of domestic animals contains mainly water soluble vitamins C and B. The role of vitamins A, D and E in the viability and fertilising ability of spermatozoa is
Ž . Ž .
not clear. Kolev 1997 examined the effects of vitamins A 100 and 200 IUrml , D
Ž200 and 400 IUrml and E 0.3 and 0.5 mg. Ž rml added to the freezing diluent for.
buffalo semen. The best post-thaw motility was obtained after addition of vitamin E at
Ž .
0.3 mgrml dose. Satisfactory motility was also obtained with vitamin A 100 IUrml . Thus, some of the vitamins may have a positive effect on post-thaw viability of buffalo spermatozoa.
Ž . Ž .
Dhami and Sahni 1993 and Dhami et al. 1994 examined the effects of raffinose
Ž1% ,. L-cysteine 0.1% and EDTA 0.1% in the generally used freezing media forŽ . Ž .
Ž .
buffalo semen tris-, milk- citrate-based diluents . The beneficial effects of raffinose on post-thaw motility and the fertility rate were observed only in the citrate buffer which did not contain other sugar.L-cysteine and EDTA improved the fertility rate, compared to raffinose or the control extenders without additive. This was attributed to significantly
Ž .
better progressive motility of spermatozoa when chelating agents cysteine and EDTA were present in the diluent. Citric-whey extender supplemented with 0.1% and 0.2%
Ž .
cysteine increased post-thaw sperm motility, particularly at the higher 0.2%
concentra-Ž .
tion Singh et al., 1990 .
Sodium pyruvate, an energy source that can be readily used by the cells, had a beneficial effect on post-thaw sperm survival only when added 1 h before freezing to
Ž .
citrate–yolk–glycerol diluent Del Sorbo et al., 1995b . Caffeine was found to stimulate
Ž .
motility of buffalo spermatozoa. El-Menoufy et al., 1985 .
5.1.3. Antibiotics
Ž . Ž .
Penicillin 1000 IUrml and streptomycin sulphate 1.0 mgrml alone or in
combina-Ž .
tion are commonly added to freezing diluents. Aleem et al. 1990 examined the microorganisms present in buffalo bull semen, and their sensitivity to commonly available antibiotics. They found that a combination of penicillin and neomycin was more effective than the combination of penicillin and streptomycin currently used.
Table 4 describes the composition of some diluents recommended for freezing buffalo semen.
5.2. Processing of semen
5.2.1. Transfer of semen samples
The breeding farms are often far from laboratories and there may be a delay before semen samples are processed. No changes have been observed in motility, morphology or freezability, of spermatozoa, if the semen was processed within 1 h of collection
ŽFabbrocini et al., 1995 . Vale et al. 1991 recommended to keep the ejaculate in its. Ž .
Table 4
Diluents for freezing buffalo semen
Ž .A Tris-based diluent recommended by Ramakrishnan and Ariff 1994Ž .
1st step 2nd step
Bidistilled water to 1000 ml to 1000 ml
Ž .B Egg yolk–galactose–cysteine diluent recommended by Del Sorbo et al. 1995bŽ .
1st step 2nd step
Penicillin G 60 mgrl 60 mgrl
Citraterhepes buffer 0.2 M — pH 6.8 to 1000 ml to 1000 ml
Ž .C Egg yolk–tes–tris–fructose–milk diluent recommended by Vale 1997Ž .
Stock solution I 36.5 ml
Ž .D Coconut-water diluent according to Vale et al. 1997Ž .
Stock solution II 93.0 ml
Glycerol 7.0 ml
Penicillin potassium G 1000 IUrml
Streptomycin sulphate 2.0 gr100 ml
Ž .
Table 4 continued
Ž .D Coconut-water diluent according to Vale et al. 1997Ž .
1st step 2nd step
from irreversibly agglutinating and also maintain the motility of spermatozoa. If some hours are needed before the beginning of semen processing, dilution of semen immedi-ately after collection with the freezing medium and subsequent storage at 58C is
Ž .
suggested Talevi et al., 1994 . These workers found that the percentage of motile spermatozoa remained consistent for up to 6 h at 58C in a diluent containing 0.3% glycerol.
5.2.2. Methods of dilution
The semen is diluted in one or two steps to a final concentration of about 100–150=106 spermrml. When the two-step method is adapted, the second diluent portion has a higher cryoprotectant concentration than the first portion. Del Sorbo et al.
Ž1994 examined both methods using tris–egg yolk-based extenders, and found that the.
Ž .
two-step method gave better results with long 6 h equilibration, while the extenders
Ž .
used for one-step dilution required shorter 2–4 h equilibration time before freezing. Addition of glycerol to semen, using a commercial milk extender and two variants, was
Ž .
examined by Fabbrocini et al. 1995 . In the first variant, the diluent used for 1st and 2nd step dilution contained 3% and 11% glycerol, respectively, and in the second variant, the glycerol concentrations were 0.3% and 14.3% in the diluent for 1st and 2nd step dilution. The second dilution was done 1 or 6 h before freezing. The only significant improvement in motility was observed when glycerol was added 1 h before freezing. Thus, it seems that addition of glycerol 1 h before freezing is the best method when a two-step dilution is adopted. Two-step dilution with Na–pyruvate was proposed
Ž .
also by Del Sorbo et al. 1995b who performed the second dilution also 1 h before freezing.
5.3. Cooling of semen
Ž .
Talevi et al. 1994 examined slow and a rapid cooling procedures: in the first case,
Ž .
Ž . Ž .
al. 1990 and Sahni and Mohan 1990 . Slow cooling procedures were tested by
Ž .
Ramakrishnan and Ariff 1994 , who cooled the semen to 58C in 45, 65 and 90 min, and
Ž .
observed no significant difference in post-thaw motility. Dhami and Sahni 1994 cooled the semen from 308C to 58C in 60 and 120 min, and found the latter more advantageous.
Ž .
In conclusion, a slow cooling procedure 0.2–0.48Crmin is recommended during pre-freezing processing of buffalo semen.
5.3.1. Equilibration of semen
There was no agreement among investigators regarding the duration of equilibration.
Ž . Ž
Some suggested short 2–4 h periods Singh et al., 1990; Dhami and Sahni, 1994; Del
. Ž . Ž
Sorbo et al., 1995b , while others recommended longer about 6 h equilibration Rao et al., 1990; Chinnaiya and Ganguli, 1990; Dhami and Kodagali, 1990; Haranath et al.,
.
1990; Talevi et al., 1994 . It is a general belief that buffalo semen should stand at 58C for not less than 2 h and no longer than 6 h before freezing.
5.4. Freezing of semen
Ž .
After equilibration, the semen packaged in mini straws 0.25 ml is frozen in liquid nitrogen vapour. Mini straws are generally used due to their cost effectiveness and
Ž .
saving of storage space. Haranath et al. 1990 found an improvement in conception rate
Ž .
for semen frozen in mini straws as compared to that frozen in medium straws 0.5 ml
Ž52.7% vs. 50.4% ..
Freezing in liquid nitrogen vapour is practical and can be done by using a simple isotherm box. The straws are suspended in horizontal position 1–4 cm above liquid nitrogen for 10–20 min, after which they are immersed into liquid nitrogen aty1968C.
Ž . Ž .
Del Sorbo et al. 1995b examined two freezing procedures: 1 a ‘‘step-wise curve’’,
Ž .
using a freezing tray Taylor Wharton , on which the straws were exposed to
tempera-Ž . Ž . Ž . Ž .
tures of q58C 4 min , y158C 7 min , y808C 15 min and y1308C 15 min in
Ž . Ž .
liquid nitrogen vapour, then immersed into liquid nitrogen see Fig. 4 ; and 2 a ‘‘continuous curve’’ that involved decreases of temperature fromq58C toy308C at a speed of 208Crmin, fromy308C toy1008C at a speed of 158Crmin, then standing for
Ž .
Ž .
Fig. 5. ‘‘Continuous’’ freezing curve according to Del Sorbo et al. 1995b .
Ž .
5 min at y1008C, before immersion into liquid nitrogen see Fig. 5 . The post-thaw recovery rates were better for ‘‘step-wise’’ than for continuous freezing curve. In the
Ž .
continuous freezing curve of Vale 1997 , the rates of decrease in temperature are
Ž .
188Crmin fromq48C toy408C and 88Crmin fromy408C to y1408C see Fig. 6 . Programmable freezers may be used to monitor the above curves.
5.5. Thawing of semen
To avoid recrystallization, rapid warming rates are commonly used. Dhami et al.
Ž1994 and Vale 1997 suggested thawing of straws in a water bath at 40. Ž . 8C for 30 s.
Ž . Ž .
Somewhat slower thawing rates were used by Kumar et al. 1993b 378C for 30 s ,
Ž . Ž . Ž . Ž
Ramakrishnan and Ariff 1994 358C for 30 s and Fabbrocini et al. 1995 398C for 30
. Ž .
s . El-Amrawi 1997 examined the effects of thawing time and of post-thaw
tempera-Ž .
ture on the quality of buffalo semen. Medium straws 0.5 ml were thawed in a water bath at 358C for 12 s or 1 min, then transferred to a water bath at 108C, 208C or 308C for 1 min, and again in a water bath at 408C for 1 min. The best post-thaw motility rate
Ž50.8% was obtained after thawing at 35. 8C for 1 min and subsequent transfer of straws to a post-thaw temperature of 108C. The thawing time had an effect on the internal temperature of the straws: straws thawed for 12 s had an internal temperature ofy48C, while those thawed for 1 min reached an internal temperature of 368C, which also
Ž .
affected the acrosome integrity. Although some workers state that the quality of buffalo semen frozen in straws can be improved by using a longer thawing time, Ziada et al.
Ž1992 found no difference between thawing at 35. 8C for 30 s and at 508C for 15 s.
5.5.1. Characteristics of thawed spermatozoa
Several parameters are used to evaluate the morphological and physiological state of spermatozoa after freeze–thawing, particularly motility, forward motility and membrane
Ž .
integrity monitored by lectins and sperm–oocyte interaction. Fabbrocini et al. 1996 examined the integrity of the plasma membrane of thawed spermatozoa using MPA-FITC lectin that binds to lectin-similar receptors on the cell surface. Changes in acrosome membrane that occur during the freeze–thawing procedure may prevent the lectin from binding to the surface sugars, and this could be considered as an evaluation parameter of non-lethal damage to the cell.
Another method for evaluation of frozen–thawed semen is the analysis of enzymes
Ž . Ž .
which may be related to fertility Table 5 . Akhtar et al. 1990 found a significant increase in hyaluronidase activity in frozen–thawed semen using the Rhodes method
ŽRhodes et al., 1971 . The activities of aspartate aminotransferase AAT , hyaluronidase. Ž .
Ž . Ž . Ž .
aminotransferase HAT , lactic dehydrogenase LDH Bhosrekar et al., 1994 , KPH
ŽKind and King, 1954 and acid phosphatase APH. Ž . ŽKing and Jaggatheesan, 1959 were.
Ž .
measured by Bhosrekar et al. 1994 . They found that the levels of all enzymes in extra-cellular medium increased significantly after freeze–thawing, which indicates leakage of enzymes from sperm cells. The release of HAT and AAT showed a highly significant negative correlation with motility and acrosomal integrity, while LDH, APH and KPH had a positive relation with motility and acrosomal integrity. The transami-nases in spermatozoa are intrinsically associated with their metabolic activity and
Ž .
function as a reservoir of energy. Kaker and Anand 1984 stressed the importance of levels of GOTrGPT enzymes in the seminal plasma as an indicator of the quality of frozen–thawed semen, since the GOTrGPT release is influenced by factors such as cold
Ž .
shock, glycerol concentration, cooling and freezing rates. Dhami and Kodagali 1990
Ž .
and Dhami and Sahni 1994 utilised the GOTrGTP, AKP, ACP and LDH levels to assess the quality of spermatozoa after freeze–thawing procedures. Fertility rates showed a high negative correlation with the release of all five enzymes, which indicates that enzyme leakage may be a marker for the assessment of freezability and fertility.
Table 5
Modification of some enzymatic activity in buffalo semen in relation to freeze–thawing process
Parameters Fresh semen Thawed semen Reference
Ž .
Hyaluronidase act. Urml 3.0 6.82 Akhtar et al., 1990
Ž .
Asp. am. transf. act. Urml 214.06 1619.18 Bhosrekar et al., 1994
Ž .
Lact. dehydr. act. Ur100 ml 5153.23 35,103.3 Bhosrekar et al., 1994
Ž .
Alk. phosp. act. Ur100 ml 83.74 726.90 Bhosrekar et al., 1994
Ž .
Acid phosp. act. Ur100 ml 58.21 521.36 Bhosrekar et al., 1994
Ž .
Ž . Ž .
Oba et al. 1994 and Vale 1997 used the thermoresistance test to evaluate the
Ž
quality of thawed semen, examining the sperm motility at different times 0, 30, 60, 120,
.
180 min during incubation at a temperature of 408C.
When frozen–thawed semen is used for in vitro insemination, in the absence of uterine or tubal fluids that facilitate motility and enhances capacitation, the spermatozoa need to be capacitated in an artificial culture medium. Different commercial culture
Ž .
media were examined by Del Sorbo et al. 1992 .
5.6. Use of frozen–thawed semen for insemination and fertility results
The fertility rate is considered to be the best parameter to assess the quality of
Ž .
frozen–thawed semen Vale, 1997 . However, a shortcoming of the assessment is that due to the high number of spermatozoa used for AI, variations in the state of sperm cells may not be reflected by the fertility rates. On the other hand, laboratory tests can indicate the extent of sperm damage during freeze–thawing, but they cannot accurately predict fertility of spermatozoa.
The difficulty regarding AI in buffaloes is the application of the method in the field and not the semen technology. The most common problems are poor conditions of
Ž .
hygiene, detection of oestrus, method and time of insemination Vale, 1997 . Detection
Ž
of oestrus in buffaloes is problematic because of the scarce behavioural signs Ohashi,
.
1994 and the muddy appearance of buffalo cows. These problems have been reported
Ž .
by authors from different countries Danell et al., 1984, Drost et al., 1985 . Zicarelli et
Ž .
al. 1997a have examined the role of teaser bulls in detection of estrus and fertility after AI. Cows run with a vasectomised bull had higher pregnancy rate than the control
Ž . Ž
females after AI in natural oestrus 42.5% vs. 18.9% and in induced oestrus of 51.1%
.
vs. 33.3% . According to the authors, buffaloes adapt with difficulty to an artificial breeding system which does not take into account their ethological needs.
The low conception rates to AI in buffaloes could also be due to the small size of the uterine body in comparison to that of bovines. Because of this, the semen could inadvertently be introduced into one uterine horn and not into the body of uterus
ŽZicarelli et al., 1997b . To clarify the situation, Zicarelli et al. 1997b compared. Ž .
cervical insemination with deposition of semen into the cranial section of the uterine horns, and obtained better fertility by the latter method when it was performed 24 h before ovulation. There was no difference between the two methods when the insemina-tions was performed after ovulation.
Ž .
Villa and Fabbri 1993 conducted two AI trials in farms located in three Italian
w Ž .
provinces using semen frozen in Laiciphos IMV extender and doses of insemination
6 Ž
containing 8=10 live spermatozoa. The high variation in the conception rates 30.5–
.
57.1% found for buffalo cows inseminated by the same method in all farms indicated a strong influence of environmental factors.
Ž . Ž .
Haranath et al. 1990 examined the efficiency of freezing in mini 0.25 ml and
Ž .
medium 0.5 ml straws using Murrah buffalo semen extended with a tris–yolk–glycerol
Ž
medium. The fertility rate was higher for packaging in mini than medium straws 52.7%
.
in tris-, citrate- and lactose-based extenders were 42.7%, 39.8% and 37.5%, respectively
ŽDhami and Kodagali, 1990 . Also in Surti buffaloes, semen frozen with a lactose–.
Ž .
yolk–glycerol extender yielded 59.1% pregnancy rate Dhami et al., 1994 .
Ž .
In a further study Dhami and Sahni, 1994 comparing different cooling rates, the
Ž .
highest fertility rate 68.1% in Murrah buffaloes was obtained for semen cooled at a
Ž .
velocity of 0.28Crmin. El-Amrawi 1997 examined the fertility of semen thawed by
Ž .
different procedures and found that the best fertility rate 64.5% for semen thawed at
Ž .
358C for 60 s. According to Vale 1997 , a pregnancy rate higher than 50% can be regarded as a good result after insemination with frozen–thawed buffalo semen.
6. Summary of procedure for freeze–thawing of semen
The procedure of processing the semen can be outlined as follows:
Ø Collection of semen by artificial vagina at 39–418C.
Ø Transfer of semen from collection to the laboratory; maximum time 6 h at 8–198C. Ø Assessment of volume, concentration, motility, forward motility, viability, acrosome
and membrane integrity of spermatozoa.
Ž
Ø Dilution of semen in one or two steps with the appropriate diluent final sperm
6 .
concentration 120–150=10 spermatozoarml .
Ø Cooling of semen to 58C at a velocity of 0.2–0.48Crmin.
Ž .
Ø Packaging of semen in straws 0.25 ml .
Ø Equilibration, 2 h for one-step dilution, 4–6 h for two-step dilution; second dilution 1 h before freezing.
Ø Freezing of straws about 4 cm above liquid nitrogen for 10–20 min, or by the freezing curves indicated and, then transfer of frozen straws into liquid nitrogen. Ø Thawing of frozen straws in a water bath at 37–458C for 15–60 s.
Ø Use of frozen–thawed semen for insemination.
Several diluents and various cooling, freezing and thawing rates have been used, at times with contrasting results. According to some authors, the freezability of buffalo spermatozoa is poor, and this is correlated to the low membrane phospholipid content and its loss during freeze–thawing.
Ž .
Finally, it is suggested to optimise adjust the phases of the freeze–thawing procedure according to the ‘‘need’’ of individual animals and the season during which the semen is collected.
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