Ž .
Animal Reproduction Science 62 2000 55–76 www.elsevier.comrlocateranireprosci
ž 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. q 2000 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 Ž
. Raizada 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 Ž
. Kumar 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: giosansocds.unina.it G. Sansone .
0378-4320r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž
. PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 1 5 4 - 8
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 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 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
. Volume ml
3 2 to 8 Ž .
Motility 70
Ž .
Vigour motility score 3
6 6
Ž .
Concentration cellsrml 600=10 to 1200=10
Ž . Live sperm
70 Ž .
Abnormal sperm -
70 pH
6.7 to 7.5
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 = 10
6
to 1290.7 100 = 10
6
cellsrml 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 = 10
6
to 1031.4 28.7 = 10
6
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
. 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 buffaloes
Ž .
Ž .
Aguiar et al., 1994 and Murrah buffaloes Kumar et al., 1993a . The latter authors 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 .
Fig. 1. Spermatozoa MPA-FITC marked: 1 Pattern 1; 2 Pattern 2 Obj. 40= .
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
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 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
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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 255.55 mmolrg, or 14.3 mmolrcell. 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 293.333.39
283.752.31 Ž
. Total proteins gr100 ml
3.100.10 2.860.14
Ž .
Total lipids mgr100 ml 321.1518.41
260.8612.52 Ž
. Fructose mgr100 ml
547.0861.24 684.6081.14
Ž .
Citric acid mgr100 ml 368.7314.82
466.3331.66 Ž
. Sodium mgr100 ml
260.638.81 258.5813.65
Ž .
Potassium mgr100 ml 153.502.68
154.833.27 Ž
. Calcium mgr100 ml
32.042.77 32.423.10
Ž .
Magnesium mgr100 ml 6.170.41
6.460.39 Ž
. Chloride mgr100 ml
196.572.45 224.062.60
Ž .
Inorganic phosphatase mgr100 ml 17.021.67
12.751.09 Ž
. Acid phosphatase Ur100 ml
225.002.99 230.461.48
Alkaline phosphatase 326.052.16
331.202.60 Ž
. Zn mmolrcell and mmolrl
14.3 86.88
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
