Storage of buffalo ž Bubalus bubalis semen · 2012. 4. 28. · Animal Reproduction Science 62 2000 55–76Ž. Storage of buffalo žBubalus bubalis/semen G. Sansone), M.J.F. Nastri,

Ž .Animal Reproduction Science 62 2000 55–76www.elsevier.comrlocateranireprosci

ž /Storage of buffalo Bubalus bubalis semenG. 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 freezingŽ .and thawing of semen are reviewed, and fertility results after artificial insemination AI with

frozen–thawed semen are given. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Buffalo; Semen; Spermatozoa; Diluent; Freezing; Thawing

1. Introduction

ŽThe 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. Recentstudies have shown that buffalo semen can be preserved just like bovine semen. Thereare a number of diluents and cryoprotectants, which are suitable for freezing buffalosemen, but there is limited information available on the semen characteristics of thespecies.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. Buffalospermatozoa are more susceptible to hazards during freezing than cattle spermatozoaŽ .Raizada et al., 1990 . These hazards can be minimised by optimising the cooling andfreezing rates and using appropriate diluting media in which spermatozoa are frozenŽ .Kumar et al., 1992a . This review deals with the characteristics, methods of processingand storage of buffalo semen.

) Corresponding author. Tel.: q39-81-552-7736; fax: q39-81-542-4848.Ž .E-mail address: [email protected] G. Sansone .

0378-4320r00r$ - see front matter q2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0378-4320 00 00154-8

( )G. Sansone et al.rAnimal Reproduction Science 62 2000 55–7656

2. Collection and characteristics of semen

Semen samples are collected from trained buffalo bulls by using an artificial vaginamaintained at temperature between 398C and 418C. The male buffalo is perhaps theeasiest domestic species to be trained to serve an artificial vagina. The semen is usuallycollected early in the morning, before feeding, and each collection consists of twoejaculates 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 abnormalŽ .spermatozoa. Den Daas 1992 reported that the relationship between the semen

characteristics and fertility has often been inconsistent. Characteristics of semen areshown in Table 1.

2.1. Colour and Õiscosity

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 inviscosity 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 amongŽ .buffaloes bred in different countries Alexiev et al., 1994; Misra et al., 1994 .

2.3. pH of semen

ŽBuffalo 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 1Ž .Characteristics of buffalo semen collected by artificial vagina according to Vale, 1994a

Characteristics

Colour white, milky white, with light blue tingeŽ . Ž .Volume ml 3 2 to 8Ž .Motility % )70Ž .Vigour motility score )3

6 6Ž .Concentration cellsrml 600=10 to 1200=10Ž .Live sperm % )70

Ž .Abnormal sperm % -70pH 6.7 to 7.5

( )G. Sansone et al.rAnimal Reproduction Science 62 2000 55–76 57

2.4. Concentration of spermatozoa

Most routine measurements of concentration are made by a spectrophotometer orŽ .haemocytometer. Woelders 1991 suggests that the fluorimetric measurement of the

amount of DNA might give a reliable estimate of concentration of spermatozoa.Ž . 6Buffalo 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 from690.6"187.9=106 to 1290.7"100=106 cellsrml in water buffaloes bred in Italy. In

Ž .Murrah buffalo bulls bred in India, Kumar et al. 1993a found sperm concentrationsfrom 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. Asmall drop of semen is placed on a dry slide maintained at 378C, and examined at amagnification of 40- or 100-fold. The Makler chamber is equipped with a grid whichfacilitates cell counting.

Ž .In the last 10 years, the introduction of computer-aided semen analysis CASA hasenabled those working in the field to use new parameters in assessing sperm motility. Ina semen sample, there can be variations in the degree of progressive movement of cellsand in lateral dislocation of sperm heads. The CASA system can evaluate parameterslike speed, direction and the beat cross frequency of sperm cells. Forward movingspermatozoa and only those which traced five straight tracks and had a minimum

Žvelocity 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 inŽ .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, thepercentage of motile spermatozoa varied from 60.8"1.5% to 69"4%, while theoccurrence of non-motile samples was about 30%. However, non-motile spermatozoaregained their motility after dilution.

2.6. Viability

The percentage of live spermatozoa determines the quality of the ejaculate. Semenwith more than 30% initial dead spermatozoa may not be suitable for storage andfreezing. 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 asŽ .head, middle-piece and tail abnormalities Kumar et al., 1993a . In semen of Nili–Ravi


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 buffaloesŽ . Ž .Aguiar et al., 1994 and Murrah buffaloes Kumar et al., 1993a . The latter authorssuggested that semen showing over 15–20% abnormal spermatozoa should be examinedfor their fertility.

2.8. Acrosomal and membrane integrity

Most workers examined the acrosome abnormalities by using the Giemsa stainŽtechnique Bhosrekar et al., 1994; Raizada et al., 1990; Ramakrishnan and Ariff, 1994;

. ŽRao 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 ofŽ .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 waterŽ .buffalo using a fluoresceinated lectin. Fabbrocini et al. 1996 used fluoresceinated

Ž .lectin, FITC-labeled Maclura pomifera Agglutinin MPA , that binds to lectin-similarreceptors 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, 2Ž .cells with the external border of the acrosome and the tail coloured, 3 uncoloured cells

Ž . Ž . Ž .Fig. 1. Spermatozoa MPA-FITC marked: 1 Pattern 1; 2 Pattern 2 Obj. 40= .


Table 2Ž . Ž .Composition of buffalo semen according to Vale 1997 mgr100 ml

Carbohydrates Fructose 623 Siliac acid 133Nitrogenous costituents Total nitrogen 684 Non-protein nitrogen 136Organic acids Citric acid 441 Ascorbic acid 6.2Inorganic costituents Total P 108, Inorganic P 5.4 Ca 44.4, Mg 4.3

Ž .see 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 semenare shown in Tables 2 and 3. Zinc is closely linked to sperm morphology, physiologyand biochemistry. The biochemical functions in which Zn has been implicated includeenzymatic function, the protein and carbohydrate metabolism and possibly spermmotility. Also, Zn concentration both in spermatozoa and seminal plasma is one of theregulating 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 seminalplasma and correlated the concentrations with the age of buffalo bulls and semenquality. No significant changes were found in the amount of zinc in spermatozoa, butthere was a marked decrease in seminal plasma with the advancement of age. The Znconcentration in seminal plasma averaged 86.88 mmolrl, whereas its concentration insperm cells was greater and averaged 255.55 mmolrg, or 14.3 mmolrcell. The increasein motility and the decrease in percentage of abnormalities are correlated to the increase

Table 3ŽPhysical and biochemical characteristics of whole semen and seminal plasma modified by Ibrahim et al.,

.1985Characteristics Whole semen Seminal plasma

Ž .Osmolarity mosMrkg 293.33"3.39 283.75"2.31Ž .Total proteins gr100 ml 3.10"0.10 2.86"0.14

Ž .Total lipids mgr100 ml 321.15"18.41 260.86"12.52Ž .Fructose mgr100 ml 547.08"61.24 684.60"81.14Ž .Citric acid mgr100 ml 368.73"14.82 466.33"31.66

Ž .Sodium mgr100 ml 260.63"8.81 258.58"13.65Ž .Potassium mgr100 ml 153.50"2.68 154.83"3.27

Ž .Calcium mgr100 ml 32.04"2.77 32.42"3.10Ž .Magnesium mgr100 ml 6.17"0.41 6.46"0.39

Ž .Chloride mgr100 ml 196.57"2.45 224.06"2.60Ž .Inorganic phosphatase mgr100 ml 17.02"1.67 12.75"1.09

Ž .Acid phosphatase Ur100 ml 225.00"2.99 230.46"1.48Alkaline phosphatase 326.05"2.16 331.20"2.60

Ž .Zn mmolrcell and mmolrl 14.3 86.88


in Zn concentration in the spermatozoa, while no relation was found between Znconcentration in the seminal plasma and motility of spermatozoa.

2.10. Coating antigens

The sperm coating antigen pattern on fresh buffalo spermatozoa was used as aŽ .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 contributionof 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 thereproductive 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 maskingŽfunction 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 reliableindication 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 asimple technique to assay the capacity of buffalo spermatozoa to bind to the zonapellucida. As buffalo oocytes are difficult to obtain, due to the fact that females of thisspecies 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 resultsshowed 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 ofŽ .spermatozoa of many mammals including buffalo provided the spermatozoa have

completed capacitation and acrosomal reaction, and can therefore be used for assessingŽ .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 sexualŽ .activity of buffalo bulls Vale, 1997 . Several authors reported that the season has an


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 theconsiderable environmental differences.In the temperate regions of the world, it has been found that the semen is of better

Žquality 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 findingduring 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

Žregard 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 .

ŽThere 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 inmotility, 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.

Ž .1993b recommended that before use for dilution, the milk should be heated, cooledovernight in a refrigerator, then the fat layer removed and the milk reheated in a waterbath for 10–12 min. After repeated cooling, the remaining fat should be removed byfiltration through cotton wool.

wŽ . xTris hydroxymethyl aminomethane , egg yolk–citrate and egg yolk–lactose are alsopopular 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

Žthree 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 trisbuffer. 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,Žglycerol was not required in the extender; but in case of a longer period of storage 72 h

.or more glycerol protected the motility of the spermatozoa. While there was nodifference between 3% and 6% glycerol for up to 24 h of storage, beyond 24 h motilityof spermatozoa was maintained better with 6% than 3% and 9% glycerol in the extender.


Ž .In studies of different egg yolk 1%, 2.5%, 5%, 10%, 20% concentrations inŽ .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 inviability of spermatozoa after 72 h stored at 58C when 5%, 10% and 20% egg yolk wasincluded 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 themedium used for dilution of semen before freezing. The diluent is usually composed of abuffer medium to which cryoprotectants and other substances are added, which protectthe 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 andlactose–egg yolk diluents did not differ in their ability to protect spermatozoa againstfreeze–thawing damages. However, the tris-based diluent yielded higher post-thaw

Ž .Fig. 2. Tris–yolk-based extender: sperm vision is not very clear Obj. 20= .


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 thanŽ .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,Žand 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,

Žand 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 thevarious extenders, which may not emerge in tests with bovine semen, indicate thatbuffalo spermatozoa are more prone to freezing stress. The poor freezability may becorrelated to the low membrane phospholipid content and to its loss during freeze–thaw-ing.

5.1.1. CryoprotectantsBoth egg yolk and glycerol are used in combination as cryoprotectants. The lecithin

and lipoprotein contents in egg yolk contribute to the preservation of the lipoproteinŽ .sheath of the sperm cell Kumar et al., 1992a . However, in addition to its protective

Ž .Fig. 3. Citrate–yolk-based extender: sperm vision is better than in the tris-based medium Obj. 20= .


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 thedialysable fraction of egg yolk and yields hydrogen peroxide, which is toxic to

Ž .spermatozoa during storage under aerobic conditions Sahni and Mohan, 1990 . For thisreason, the egg yolk may be dialysed before addition to the extender. Little attention hasbeen paid to the level of egg yolk necessary for freezing buffalo semen, and the majorityof workers are using concentrations around 20%. It should be noted, however, that as theyolk concentration is increased in the diluent, the pH of the medium decreases and tendstowards the acidic side. This may also be the reason for the depressing effect of higheramounts 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 significantimprovement in post-thaw motility. This indicated the scope of reducing the yolk levelfrom 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 gaveprotection to spermatozoa during freeze–thawing. After addition of egg yolk, post-thawmotility 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 itwith 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 testedŽ .Kumar 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 atŽ .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. Inmilk diluent, fructose and sucrose gave the best protection, as judged by post-thawmotility results, due probably to the synergy action of lactose present in milk. Someprotection 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 acryoprotective role by altering the permeability of the cell membrane and by maintainingthe electrolyte balance. However, the best post-thaw motilities were never higher than30% 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 tohave properties that protect bovine and caprine spermatozoa. Coconut water has beenshown to be an alternative for storage of bovine semen, as the phytohormones it contains

Žprotect 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 coconutŽ .water-based diluent for processing buffalo semen was shown by Vale et al. 1997 . The

coconut water obtained from fresh fruit must be filtered and, at its final state before usefor dilution, should have an osmolarity of 320 mosM and pH 6.8.


5.1.2. AdditiÕes to freezing diluentsMany 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 isŽ . Ž .not clear. Kolev 1997 examined the effects of vitamins A 100 and 200 IUrml , D

Ž . Ž .200 and 400 IUrml and E 0.3 and 0.5 mgrml added to the freezing diluent forbuffalo 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 buffalospermatozoa.

Ž . Ž .Dhami and Sahni 1993 and Dhami et al. 1994 examined the effects of raffinoseŽ . Ž . Ž .1% , L-cysteine 0.1% and EDTA 0.1% in the generally used freezing media for

Ž .buffalo semen tris-, milk- citrate-based diluents . The beneficial effects of raffinose onpost-thaw motility and the fertility rate were observed only in the citrate buffer whichdid not contain other sugar. L-cysteine and EDTA improved the fertility rate, comparedto raffinose or the control extenders without additive. This was attributed to significantly

Ž .better progressive motility of spermatozoa when chelating agents cysteine and EDTAwere 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 toŽ .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. AntibioticsŽ . 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 themicroorganisms present in buffalo bull semen, and their sensitivity to commonlyavailable antibiotics. They found that a combination of penicillin and neomycin wasmore 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 samplesThe 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, morphologyor freezability, of spermatozoa, if the semen was processed within 1 h of collectionŽ . Ž .Fabbrocini et al., 1995 . Vale et al. 1991 recommended to keep the ejaculate in itsown plasma for 10–15 min, although semen of some buffaloes may show agglutination.In this case, addition of the diluent immediately after collection could prevent the semen


Table 4Diluents for freezing buffalo semenŽ . Ž .A Tris-based diluent recommended by Ramakrishnan and Ariff 1994

1st step 2nd step

Egg yolk 20% 20%Tris 3.02% 3.02%Citric acid 1.67% 1.67%Ž .D y Fructose 1.25% 1.25%

Glycerol 4% 12%Penicillin 1000 IUrml 1000 IUrmlStreptomycin 1.0 mgrml 1.0 mgrmlBidistilled water to 1000 ml to 1000 ml

Ž . Ž .B Egg yolk–galactose–cysteine diluent recommended by Del Sorbo et al. 1995b

1st step 2nd step

Egg yolk 20% 20%Ž .D y galactose 0.5% 0.5%

Cysteine 6.4 mM 6.4 mMCaffeine 7 mM 7 mMVitamin C 0.7 mM 0.7 mMNa-pyruvate – 2.5 mMGlycerol 0.3% 12.3%Penicillin G 60 mgrl 60 mgrlCitraterhepes buffer 0.2 M — pH 6.8 to 1000 ml to 1000 ml

Ž . Ž .C Egg yolk–tes–tris–fructose–milk diluent recommended by Vale 1997

Stock solution I 36.5 mlStock solution II 36.5 mlGlycerol 7.0 mlEgg yolk 7.0 mlŽ .Adjust pH to 6.8–7.0 with NaOH 10%

Stock solution ITes 48.3 gTris 11.6 gŽ .D y Fructose 2.0 g

Bidistilled water 1000 ml6Penicillin G 1=10 IU

Streptomycin sulphate 2.0 g

Stock solution IISkim milk 110.0 gBidistilled water 1000 mlŽ .Heat at 928C for 5 min

Ž . Ž .D Coconut-water diluent according to Vale et al. 1997

Stock solution II 93.0 mlGlycerol 7.0 mlPenicillin potassium G 1000 IUrmlStreptomycin sulphate 2.0 gr100 mlŽ .Adjust pH to 6.8–7.0 with NaOH 10%


Ž .Table 4 continuedŽ . Ž .D Coconut-water diluent according to Vale et al. 1997

1st step 2nd step

Stock solution ICoconut water 50.0 mlBidistillated water 25.0 mlSodium citrate 5% 25.0 mlq.s.p. 25.0 ml

Stock solution IIStock solution I 90.0 mlEgg yolk 10.0 mlq.s.p. 100.0 ml

from irreversibly agglutinating and also maintain the motility of spermatozoa. If somehours 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 motilespermatozoa remained consistent for up to 6 h at 58C in a diluent containing 0.3%glycerol.

5.2.2. Methods of dilutionThe 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 diluentportion has a higher cryoprotectant concentration than the first portion. Del Sorbo et al.Ž .1994 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 extendersŽ .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, wasŽ .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 secondvariant, the glycerol concentrations were 0.3% and 14.3% in the diluent for 1st and 2ndstep dilution. The second dilution was done 1 or 6 h before freezing. The onlysignificant improvement in motility was observed when glycerol was added 1 h beforefreezing. Thus, it seems that addition of glycerol 1 h before freezing is the best methodwhen 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 beforefreezing.

5.3. Cooling of semen

Ž .Talevi et al. 1994 examined slow and a rapid cooling procedures: in the first case,Ž .the semen having an initial temperature of 288C reached a temperature of 58C in 1 h, in

the second case in 15 min. There was no significant difference in post-thaw motility ofspermatozoa cooled rapidly or slowly. Similar cooling rates were also used by Singh et


Ž . Ž .al. 1990 and Sahni and Mohan 1990 . Slow cooling procedures were tested byŽ .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 cooledthe 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 duringpre-freezing processing of buffalo semen.

5.3.1. Equilibration of semenThere was no agreement among investigators regarding the duration of equilibration.

Ž . ŽSome suggested short 2–4 h periods Singh et al., 1990; Dhami and Sahni, 1994; Del. Ž . ŽSorbo 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 liquidnitrogen 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 rateŽ .for semen frozen in mini straws as compared to that frozen in medium straws 0.5 ml

Ž .52.7% 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 liquidnitrogen for 10–20 min, after which they are immersed into liquid nitrogen at y1968C.

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 inŽ . Ž .liquid nitrogen vapour, then immersed into liquid nitrogen see Fig. 4 ; and 2 a

‘‘continuous curve’’ that involved decreases of temperature from q58C to y308C at aspeed of 208Crmin, fromy308C toy1008C at a speed of 158Crmin, then standing for

Ž .Fig. 4. ‘‘Step-wise’’ freezing curve according to Del Sorbo et al. 1995b .


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-thawrecovery 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 areŽ .188Crmin from q48C to y408C and 88Crmin from y408C 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.Ž . Ž .1994 and Vale 1997 suggested thawing of straws in a water bath at 408C for 30 s.

Ž . Ž .Somewhat slower thawing rates were used by Kumar et al. 1993b 378C for 30 s ,Ž . Ž . Ž . ŽRamakrishnan 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 for1 min, and again in a water bath at 408C for 1 min. The best post-thaw motility rateŽ .50.8% was obtained after thawing at 358C for 1 min and subsequent transfer of strawsto a post-thaw temperature of 108C. The thawing time had an effect on the internaltemperature of the straws: straws thawed for 12 s had an internal temperature of y48C,while those thawed for 1 min reached an internal temperature of 368C, which also

Ž .Fig. 6. ‘‘Continuous’’ freezing curve according to Vale et al. 1997 .


affected the acrosome integrity. Although some workers state that the quality of buffalosemen frozen in straws can be improved by using a longer thawing time, Ziada et al.Ž .1992 found no difference between thawing at 358C for 30 s and at 508C for 15 s.

5.5.1. Characteristics of thawed spermatozoaSeveral parameters are used to evaluate the morphological and physiological state of

spermatozoa after freeze–thawing, particularly motility, forward motility and membraneŽ .integrity monitored by lectins and sperm–oocyte interaction. Fabbrocini et al. 1996

examined the integrity of the plasma membrane of thawed spermatozoa using MPA-FITClectin that binds to lectin-similar receptors on the cell surface. Changes in acrosomemembrane that occur during the freeze–thawing procedure may prevent the lectin frombinding to the surface sugars, and this could be considered as an evaluation parameter ofnon-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 significantincrease in hyaluronidase activity in frozen–thawed semen using the Rhodes methodŽ . Ž .Rhodes et al., 1971 . The activities of aspartate aminotransferase AAT , hyaluronidase

Ž . Ž . Ž .aminotransferase HAT , lactic dehydrogenase LDH Bhosrekar et al., 1994 , KPHŽ . Ž . Ž .Kind and King, 1954 and acid phosphatase APH King and Jaggatheesan, 1959 were

Ž .measured by Bhosrekar et al. 1994 . They found that the levels of all enzymes inextra-cellular medium increased significantly after freeze–thawing, which indicatesleakage of enzymes from sperm cells. The release of HAT and AAT showed a highlysignificant negative correlation with motility and acrosomal integrity, while LDH, APHand 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 oflevels of GOTrGPT enzymes in the seminal plasma as an indicator of the quality offrozen–thawed semen, since the GOTrGPT release is influenced by factors such as cold

Ž .shock, glycerol concentration, cooling and freezing rates. Dhami and Kodagali 1990Ž .and Dhami and Sahni 1994 utilised the GOTrGTP, AKP, ACP and LDH levels to

assess the quality of spermatozoa after freeze–thawing procedures. Fertility ratesshowed a high negative correlation with the release of all five enzymes, which indicatesthat enzyme leakage may be a marker for the assessment of freezability and fertility.

Table 5Modification of some enzymatic activity in buffalo semen in relation to freeze–thawing processParameters Fresh semen Thawed semen Reference

Ž .Hyaluronidase act. Urml 3.0 6.82 Akhtar et al., 1990Ž .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., 1994Ž .Alk. phosp. act. Ur100 ml 83.74 726.90 Bhosrekar et al., 1994Ž .Acid phosp. act. Ur100 ml 58.21 521.36 Bhosrekar et al., 1994

Ž .GOTrGPT release mMrl 14.94 24.11 Dhami and Sahni, 1993


Ž . Ž .Oba et al. 1994 and Vale 1997 used the thermoresistance test to evaluate theŽquality 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 spermatozoaneed 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 ofŽ .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 cellsmay not be reflected by the fertility rates. On the other hand, laboratory tests canindicate the extent of sperm damage during freeze–thawing, but they cannot accuratelypredict 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 ofŽ .hygiene, detection of oestrus, method and time of insemination Vale, 1997 . Detection

Žof 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 etŽ .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 controlŽ . Žfemales 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 artificialbreeding 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 couldinadvertently be introduced into one uterine horn and not into the body of uterusŽ . Ž .Zicarelli et al., 1997b . To clarify the situation, Zicarelli et al. 1997b comparedcervical insemination with deposition of semen into the cranial section of the uterinehorns, and obtained better fertility by the latter method when it was performed 24 hbefore 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 Italianw Ž .provinces using semen frozen in Laiciphos IMV extender and doses of insemination

6 Žcontaining 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–glycerolŽmedium. The fertility rate was higher for packaging in mini than medium straws 52.7%

.vs. 50.4% . The pregnancy rates in Surti buffaloes, after insemination with semen frozen


in tris-, citrate- and lactose-based extenders were 42.7%, 39.8% and 37.5%, respectivelyŽ .Dhami 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, theŽ .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 byŽ .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 beregarded 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, acrosomeand membrane integrity of spermatozoa.

ŽØ Dilution of semen in one or two steps with the appropriate diluent final sperm6 .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 1h before freezing.

Ø Freezing of straws about 4 cm above liquid nitrogen for 10–20 min, or by thefreezing 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, attimes with contrasting results. According to some authors, the freezability of buffalospermatozoa is poor, and this is correlated to the low membrane phospholipid contentand its loss during freeze–thawing.

Ž .Finally, it is suggested to optimise adjust the phases of the freeze–thawingprocedure according to the ‘‘need’’ of individual animals and the season during whichthe semen is collected.

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