14 Applied Andrology in Water Buffalo Sayed Murtaza H. Andrabi* Buffalo are known for their habit of wallowing in water, especially during the hot hours of the day, and hence they are also referred to as water buffalo (hereafter mostly referred to as buffalo). The domestic buffalo (Bubalus bubalis) has been broadly classified on the basis of habitat as river and swamp types, each considered a subspecies. This characteristic is so marked that the buffalo can be described as a semi-aquatic mammal. The river buffalo (B. b. bubalis) prefers to immerse itself in running water or ponds. The swamp buffalo (B. b. carabanesis) likes stagnant water or mud and prepares its own wallow by digging with its horns. Keeping buffalo away from water makes them more susceptible to environmental stresses (heat) and results in poor productive and reproductive performance (Marai and Haeeb, 2010). The world buffalo population is increasing, and in 2007 was estimated to be over 177 million head (FAO, 2007). Since 1981/2, buffalo numbers have increased by 50 million. Interest in buffalo rearing and breeding is growing in several countries where the animal has previously been neglected – and even unknown. More than 97% of the population is located in Asia, where buffalo play a prominent role in rural livestock production both providing milk and meat, and being used as working animals. In recent decades, buffalo farming has expanded widely in Mediterranean areas and in Latin America. For the manufacture of specific dairy products such as Mozzarella cheese, buffalo are reared in different regions of Australia. Buffalo are also reared for meat purposes in non-traditional countries because of the good dietary value of the meat, which contains less saturated fat than beef and pork. Only in India and Pakistan are there well-defined buffalo breeds (Drost, 2007). Here, they are classified into five major groups: the Murrah, Gujarat, Uttar Pradesh, Central Indian and South Indian breeds. The Nili-Ravi buffalo, belonging to the Murrah group, is recognized as the highest milk-producing breed of buffalo (Cockrill, 1974). The swamp buffalo found in South-east and Far East Asia has low milk production, and is mostly used as a draft animal by small farm holders or for meat purposes (Andrabi, 2009). The male plays an important role in any successful reproductive management programme. With the development of frozen semen technology for buffalo breeding, the demand for the best males has increased considerably. The aim of this chapter is to describe the major reproductive and andrological parameters of water buffalo bulls. Generally, puberty in a bull occurs when the bull calf produces sufficient sperm to successfully impregnate a female; this has been defined as the time when a bull first produces an ejaculate containing 50 million spermatozoa, of which more than 10% are progressively motile (Wolf et al., 1965). The same principle has been used to define puberty in buffalo bulls (Ahmad et al., 2010). The pubertal period is associated with rapid testicular growth, changes in luteinizing hormone (LH) release pattern, an increase in blood plasma testosterone and the initiation of spermatogenesis. The onset of puberty in buffalo bulls occurs later than it does in Bos taurus bulls. Sexual maturity of the male buffalo is attained at about 2–3 years of age, depending on the type of breed, management practices and feeding. Appropriate feeding and management of prepubertal buffalo bulls is thought to be of value in enhancing puberty, because the first signs of sexual interest and meiotic divisions of spermatogonial cells have been found to occur as early as 9 months of age (Ali et al., 1981). The first sexual interest of a buffalo bull may coincide with the development of fertilizing capacity or may precede it by a variable period. Although bulls are fertile during the immediate post-pubertal period, the quality and quantity of semen produced increases over the subsequent months. The testis size shows a curvilinear increase in relation to age. It increases slowly between 5 and 15 months of age, rapidly between 15 and 25 months of age and slowly again between 25 and 38 months of age. The plasma testosterone concentrations are low up to 21 months of age, start to rise at 25 months and reach peak levels at 38 months of age (Ahmad et al., 1984). Changes in interstitial cells during the development of the buffalo testis reveal that adult Leydig cells are visible in 3-month-old buffalo calves, but mesenchymal cells are seen from 18 months of age onwards. The percentage of adult Leydig cells reaches a maximum by 72 months of age and beyond (Rana and Bilaspuri, 2000). Some of the salient characteristics linked to puberty in buffalo bulls are presented in Table 14.1. The ages and body weights at puberty in male buffalo are quite variable, with pubertal characteristics determined more by body weight than by age. Under good conditions, testicular spermatogenic cell divisions commence by approximately 12 months and active spermatogenesis can be seen by 15 months of age (Azmi et al., 1990). However, the ejaculate contains viable spermatozoa only after 24–30 months of age, indicating that male buffaloes mature more slowly and have a longer time lag between the onset of spermatogenesis and the achievement of puberty than B. taurus bulls (Perera, 1999). Buffalo bulls are capable of breeding throughout the year, but some seasonal fluctuation in reproductive functions is evident in most countries. Heuer et al. (1987) attributed 40% of the observed seasonality of buffalo fertility to the male. In river type buffalo, semen volume, sperm concentration and initial sperm motility did not differ significantly between different seasons, but there was a significant difference post freezing (Tuli and Singh, 1983). Several studies have reported better freezability and conception rate of spermatozoa harvested during the autumn/winter or peak breeding season compared with those collected and processed during the summer (dry or wet) or low breeding season (Tuli and Singh, 1983; Heuer et al., 1987; Bhavsar et al., 1989; Sagdeo et al., 1991; Bahga and Khokar, 1991; Younis et al., 1998; Koonjaenak et al., 2007b,c). There is a possibility that a seasonal variation in the biochemical composition of seminal plasma and/or spermatozoa may occur, as it does in other farm animals (Cabrera et al., 2005; Argov et al., 2007; Koonjaenak et al., 2007c). There are a few scattered reports available that describe differences in chemical composition of buffalo seminal plasma and spermatozoa under different climatic conditions (Singh et al., 1969; Mohan et al., 1979; Sidhu and Guraya, 1979), but the information given in these studies is insufficient to explain the variation in the freezability of buffalo spermatozoa during different seasons (Andrabi, 2009). Table 14.1. Puberty in water buffalo. The male reproductive system can be divided into three components: the primary sex organ, i.e. testes; a group of accessory glands and ducts, i.e. the epididymis, vesicular glands, bulbourethral gland and prostate; and the external genitalia or copulatory organ i.e. the penis. The reproductive organs of the water buffalo bull are similar to those of the bull of domestic cattle (B. indicus), but the testes and scrotum are smaller and the penile sheath is less pendulous. The sheath of the penis adheres close to the body in the swamp type of buffalo. but is more pendulous in the river type. As in cattle, the testis and epididymis can be palpated through the scrotal wall, while the prostate, seminal vesicles and ampullae of the ductus deferens can be palpated per rectum (Ahmad and Noakes, 2009). The scrotum of the river buffalo is 20–25 cm in length, has a distinct neck and is pendulous. The scrotum of the swamp buffalo is about 10 cm in length (fully extended) and the neck is not distinct (MacGregor, 1941). The testes of the swamp buffalo descend into the scrotum at 2–4 or 6 months of age, while they may be present in the scrotum at birth in the river type. The normal testes of buffalo are ovoid in shape and turgid on palpation, with a marked resonance. The testes are of unequal size, with the left usually bigger. In river buffalo, the average measurements (length × width) of the right and left testicles (including the epididymis) are 14.2 × 6.41 cm and 15.0 × 6.87 cm, respectively. Joshi et al. (1967) reported the length, breadth and circumference of the testes, excluding the epididymis, as 7.60 × 4.30 × 12.20 cm for the right and 7.87 × 4.33 × 12.29 cm for the left. In swamp buffalo, MacGregor (1941) reported that the average right and left testicle measurements, including the epididymis length × width) were 11.18 × 4.85 cm and 11.27 × 4.82 cm, respectively. The diameter of the seminiferous tubule in water buffalo testes ranges from 170 to 200 μm and each is 5–100 cm in length. The total length is as high as 5000 m in a pair of testes. The height of the seminiferous epithelium is about 56 μm and the interstitial nuclear diameter is close to 13 μm (Ahmad et al., 2010). During calfhood, the penis is firmly adhered within the sheath and cannot be extended. However, at about 2 to 4 months before puberty, partial protrusion occurs during mounting, followed by separation of penis from the sheath, complete erection and eventually mating and ejaculation. In river buffalo, the penis hangs clear of the abdomen by 15–30 cm, being attached thereto by a triangular fold of skin running backwards from the umbilicus. In swamp type buffalo, the sheath of the penis adheres closely to the abdomen except for the last 2–3 cm (MacGregor, 1941). The average length of the penis is 83.51 cm in the river buffalo and 56.72 cm in the swamp type (Joshi et al., 1967). The prepuce of water buffalo is long and narrow, and is devoid of hairs. Libido is the eagerness of the male animal to copulate with the female. Bull libido, or sex drive, is an important aspect of fertility and there is great individual variation in it. There is a large genetic component to libido (Chenoweth, 1997) although, within bulls, Landaeta-Hernandez et al. (2001) reported poor repeatability in test results for libido, service rate and reaction time to service, suggesting that there are other influences on mating behaviour as well, such as learning and/or environmental factors. The sexual behaviour of the water buffalo bull is similar to, but less intense than, that of the B. taurus bull. Libido is suppressed during the hotter part of the day. Sniffing of the vulva or of female urine and the flehmen reaction precede mounting of the oestrous female. The occurrence of flehmen behaviour in the buffalo bull is significantly increased during oestrus compared with dioestrus (Rajanarayanan and Archunan, 2004). Mating is brief and lasts only a few seconds, and the ejaculatory thrust is less marked than in the cattle bull. After ejaculation, in contrast to the B. taurus bull, the buffalo bull dismounts slowly and the penis retracts gradually into the sheath (Jainudeen and Hafez, 1987). Following ejaculation and dismounting, the buffalo bull shows a sexual refractory period, but a quick return to mounting behaviour is shown by males when given an opportunity to mate a new oestrous female (Jainudeen and Hafez, 1987). Buffalo bulls usually continue to tease the same female buffalo and repeatedly mount her, perhaps within a 10–15 min period, but the interval between and number of mountings vary between males. Good libido and proper mating ability of a buffalo bull are also desirable traits for a successful artificial insemination (AI) programme to harvest maximum semen of acceptable quality. Wide variation in libido and sexual behaviour exists between semen donor buffalo bulls (Anzar et al., 1993). Bulls of the best category in terms of libido and sexual behaviour must be used to overcome the two different types of infertility related to these behavioural characteristics – delayed puberty and failure of semen production (Ahmad et al., 1985). Ejaculation time in seconds, one of the indices of libido, is higher in swamp buffalo than in river buffalo, 467.24 ± 353.9 and 112.57 ± 62.4, respectively (± SE) (Ramakrishnan et al., 1989). The influence of environment (season) on libido score (0–6) in river buffalo bulls was significantly higher during the autumn/early winter peak breeding season, 4.30 ± 0.13 (± SE), than in the summer low breeding season, 3.46 ± 0.16 (± SE); similarly, reaction time (s) was significantly lower, 31.24 ± 2.27 (± SE), during the autumn peak breeding season than in the summer low breeding season, 34.28 ± 3.3 (± SE) (Younis et al., 2003). Among farm animals (with the exception of the boar), the water buffalo bull has one of the shortest spermatogenic cycles. Buffalo sperm-atogenesis constitutes 4.57 cycles of the seminiferous epithelium (Guraya and Bilaspuri, 1976). The duration of the seminiferous epithelial cycle and of spermatogenesis are 8.6–8.7 days and 38 days, respectively (Sharma and Gupta, 1980; McCool et al., 1989). In general, the frequency of cell stages in water buffalo and cattle is similar, at eight (Pawar and Wrobel, 1991). In sexually mature river buffalo, the total epididymal sperm reserve per animal is about 36.2 billion. The efficiency of sperm production averages 14.5 × 106 sperm/g testicular parenchyma daily, with a mean of 2.02 × 109 sperm/testis. Thus, a typical buffalo bull produces about 4 × 109 sperm daily (Sharma and Gupta, 1979). This may vary according to age, season and nutrition. Pant et al. (2003) reported 5.2–8.4 × 106 sperm/g of testicular parenchyma daily, with a range of 2.18–3.37 × 109 total sperm daily in river buffalo bulls. In a mature swamp buffalo bull, daily sperm production is about 1.86 × 109 and epididymal sperm reserves are 9.7 × 109 (McCool and Entwistle, 1989). The overall short duration of spermatogenesis, the smaller testicular size and low rate of daily sperm production in both types of water buffalo, compared with cattle, perhaps reflects species difference in the length of the sexual season and mating behaviour (Jainudeen and Hafez, 1987). Dhingra and Goyal (1975) explained in detail the different types of spermatogenic cells in the adult water buffalo bull. Type A spermatogonia have a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm, and have one or two nucleoli adhering to the nuclear membrane. Type A0 spermatogonia are characterized by nuclei containing finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, in contrast, have finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resemble those of type A1, but contain coarse granular chromatin dispersed in the nucleoplasm. The intermediate type of spermatogonia acquire a central position of the nucleolus, but the chromatin remains coarsely granulated and non-clumped. Three classes of type B (B1–B3) spermatogonia are present based on the degree of clumping of the chromatin and the central position of the nucleolus. Type B1 cells are characterized by nuclei containing a few flakes of chromatin and a centrally located nucleolus. Type B2 cells show comparatively more clumping of chromatin than type B1 spermatogonia, and this is dispersed at random in the nucleoplasm and along the nuclear envelope. Type B3 spermatogonia show chromaphilic chromatin dispersed in the nucleoplasm and adhering along the nuclear membrane. The procedure for the collection of buffalo semen is by using a teaser and an artificial vagina (AV) (Fig. 14.1). Buffalo bulls are considered among the easiest of mammals to be trained to serve an AV (Presicce, 2007), provided that the temperature (39–42°C), type of inner liner (smooth or rough) and air pressure of the AV are appropriate. Buffalo bulls are less choosy than cattle bulls about the teaser or dummy animal and quickly mount a teaser or a male buffalo in their service of an AV for semen collection. Successful electroejaculation (EE) and transrectal message methods have also been used for semen collection in buffalo bulls. However, the quality of semen collected is better from using an AV than from EE or transrectal message. In normal routine, a 30.48 cm long bovine ‘Danish model’ AV is used for semen collection in buffalo bulls. Adult bulls prefer a smooth inner lining to the AV, whereas senile bulls donating semen prefer an AV with rough inner lining. Two consecutive ejaculates at 10–20 min interval are collected from buffalo bulls twice a week with an AV. The semen is collected after at least two false mounts (sexual stimulation). Frequent or delayed collections result in poor semen quality (Anzar et al., 1988). Buffalo bulls normally produce greyish to milky white or creamy white semen, with a slight tinge of blue (Jainudeen and Hafez, 1987; Vale, 1994). The yellowish semen produced by some Bos bulls because of the presence of riboflavin is rarely produced by buffalo bulls, although under some pathological conditions, buffalo bulls can produce semen of differing colour and consistency. The volume of the buffalo ejaculate varies from bull to bull and within each bull, depending on breed and age of bull. Significant individual variations in the semen volume of buffalo bulls have been reported by numerous workers (Table 14.2). Young bulls coming into service produce about 1–2 ml of semen, while older bulls provide up to 6 ml (Vale, 1994). In general, the volume of the semen increases with age, but it also depends upon the general and reproductive health of the animal and the frequency of ejaculation. Fig. 14.1. Semen collection from a water buffalo bull, which shows bull dismounting after thrusting. Table 14.2. Semen characteristics of water buffalo. Marked variations in the concentration of buffalo bull spermatozoa in semen have been reported (Table 14.2), with the number of spermatozoa/ml ejaculate varying from 0.2 to over 3 billion. Factors such as sexual development, age, season, nutrition and reproductive health affect the concentration of spermatozoa in ejaculates. Viscosity is a measure of the fluidity or density of the semen. The viscosity of buffalo semen is reported to be 1.88 centipoise (0.188 Pa/s). A positive correlation between viscosity and concentration has been reported for buffalo bull semen. The density of the semen varies from 0.84 to 1.12 g/ml, and is largely due to the sperm concentration. The average value of the semen surface tension is reported to be 65.03 dyne/cm (650.03 μN/cm). Sexual development, age, season, nutrition and reproductive health all affect the concentration of spermatozoa in ejaculates. Viscosity, density and surface tension of the semen, like sperm concentration, depend upon sexual development, age, season, nutrition and reproductive health (Sidhu and Guraya, 1985, pp. 8–10). Generally, the motility of buffalo bull semen is lower than that of the Bos bull. The range of sperm motility found in buffalo bull semen is shown in Table 14.2. Various sperm morphometric indices of buffalo bull spermatozoa are presented in Table 14.3. Table 14.3. Sperm morphometric indices for water buffalo. Values are either average or mean ± SEM. Buffalo semen contains higher concentrations of fructose, acid and alkaline phosphatase activity and inorganic phosphorus, but lower concentrations of citric acid and ascorbic acid than cattle bull semen. Also in contrast to cattle bull semen, higher alkaline phosphatase in buffalo semen is concomitant with decreased motility and percentage of live cells, depressed dehydrogenase activity and a slight decrease in the rate of fructose hydrolysis. Data on the enzymic activity and other biochemical constituents of water buffalo semen are given in Tables 14.4 and 14.5. Both acetylcholinesterase and amylase are significantly lower in buffalo than in cattle bull semen. The buffalo sperm acrosome is rich in hydrolytic enzymes, such as alkaline phosphatase and beta-glucuronidase, although acid phosphatase is localized mainly in the post-acrosomal segment. Buffalo semen contains characteristically low levels of potassium compared with sodium. The sodium: potassium ratio in buffalo seminal plasma is 1:3.7, on average, with well over 60% of ejaculates having a ratio of 1:2.86. The zinc (Zn) concentration in seminal plasma averages 86.88 mmol/l, whereas its concentration in sperm is greater, averaging 14.3 mmol/cell. Increased motility and a decreased percentage of abnormalities are correlated with an increased Zn concentration in spermatozoa, but no relationship has been found between Zn concentration in the seminal plasma and sperm motility.
National Agricultural Research Centre, Park Road, Islamabad, Pakistan
Introduction
Puberty
Seasonality
Genitalia
Sexual Behaviour and Libido
Sperm Production
Semen Collection
Semen Characteristics
Semen colour
Ejaculate volume
Semen pH and buffering capacity
Sperm concentration
Semen viscosity
Sperm motility
Sperm biometry
Biochemical Composition of Semen
Characteristic | Seminal plasma | Spermatozoa |
Acid phosphatase | 315.31 ± 22.66 BUa/100 ml | – |
| 194 ± 10 BU/100 ml | 39 ± 6 BU/1011 cell |
Aldolase | 70.31 ± 27.79 SLUb/ml | – |
Alkaline phosphatase | 312.50 ± 24.04 BU/100 ml | – |
| 270 ± 9 BU/100 ml | 63 ± 6 BU/1011 cell |
Deoxyribonuclease | – | 2007.33 ± 112.01 KUc/ml |
Glutamic-oxaloacetic transaminase | 166.72 ± 14.08 Ud/ml | – |
Glutamic-pyruvic transaminase | 134.56 ± 4.57 U/ml | – |
Lactic dehydrogenase | 1671.5 ± 113.11 BBUe/ml | – |
Values are either mean ± SD or mean ± SE.
Units of enzyme activity: aBU, Bodansky units; bSLU, Sibley–Lehninger units; cKU, Kunitz units;dU, (standard) units;eBBU, Berga-Broida units.
Table 14.5. Biochemical composition of water buffalo semen. Modified from Andrabi, 2009.
Characteristic | Seminal plasma | Spermatozoa |
Alanine | 0.413 mM | – |
Ascorbic acid | 3.9 ± 0.5 mg/100 ml | – |
Aspartic acid | 0.395 mM | – |
Cholesterol | 18.5 ± 0.9 % of total neutral lipids | 117.6 ± 1.6 % of total neutral lipids |
| 53.67 ± 8.72 mg/100 ml |
|
Citric acid | 444.9 ± 17.4 mg/100 ml | – |
Fructose | 368.12–815.71 mg/100 ml | – |
Glutamic acid | 4.28 mM | – |
Glutathione | 32.49 ± 5.10 μmol/ml | – |
Glycine | 1.34 mM | – |
Glycolipids | 0.581 mg/ml | 0.397 mg/109 cells |
Lactic acid | 82 ± 6 mg/100 ml | 167 ± 9 μg/1011 cells |
Lipids | 1.5 mg/ml or 1.75 ± 0.03 mg/ml | 1.320 ± 0.030 mg/109 cells |
Lysine | 0.133 mM | – |
Neutral lipids | 0.439 mg/ml | 0.286 mg/109 cells |
Phospholipids | 0.594 mg/ml | 0.548 mg/109 cells |
Serine | 0.60 mM | – |
Values are either average or mean ± SEM.
Preservation of Semen
Liquid storage
Buffalo semen can be stored at a refrigeration temperature of 5°C for up to 72 h without significant decrease in quality, provided that it is diluted in milk-based extenders or with media that have the same composition as those used for deep freezing (except for glycerol) (Dhami et al., 1994; Akhter et al., 2008, 2011b).
Fresh cow’s milk is widely used as a diluent for the liquid storage of buffalo semen (Kumar et al., 1993a). It is recommended that, before use for dilution, the milk should be heated, cooled overnight in a refrigerator, the fat layer removed and the milk then reheated in a water bath for a few minutes. After repeated cooling, the remaining fat should be removed by filtration through cotton wool. Tris(hydroxymethyl)aminomethane, egg yolk-citrate and egg yolk-lactose are also popular diluting media for the liquid storage of buffalo semen (Akhter et al., 2011b). After storage of semen in milk, Tris and citrate-based diluents at 5°C for 24 h, the decrease in motility was similar in the three media, but after 48 h, only milk and Tris were able to maintain sperm motility (Kumar et al., 1992). Dhami et al. (1994) examined the relative efficacy of Tris-, citrate- and lactose-based diluents, and found that the best sperm survival after 72 h at 5°C was in Tris buffer. Recently, Bioxcell has been found to be suitable for the liquid storage of cooled buffalo semen for up to 5 days (Akhter et al., 2011b), although fertility results are not yet available on the use of Bioxcell as a medium for this purpose.
The proportion of motile spermatozoa studied at different egg yolk concentrations (1, 2.5, 5, 10 and 20%) in Tris-based diluent showed that low levels (1 and 2.5% egg yolk) are the best for 72 h storage at 5°C (Sahni and Mohan, 1990a,b). There was no improvement in viability of spermatozoa after 72 h storage at 5°C when 5, 10 and 20% egg yolk was included in milk- or Tris-based extenders. However, there was an improvement in Tris-based extender (10 or 20% egg yolk) after the addition of 0.1% L-cysteine, or of EDTA or its tetrasodium salt (Kumar et al., 1993a).
The examination of different glycerol concentrations (3, 6 or 9%) in milk-, Tris- and citrate-based extenders (Kumar et al., 1992) revealed that for 24 h storage at 5°C, glycerol was not required in the extender; but in the case of a longer period of storage (72 h or more), glycerol protected the motility of the spermatozoa. There was no difference between 3 and 6% glycerol for up to 24 h of storage, but beyond 24 h, the motility of spermatozoa was maintained better with 6% than with 3 or 9% glycerol in the extender.
Frozen storage
Buffer
The dilution of semen in a suitable buffer is one of the important factors affecting sperm survival during cryopreservation (Rasul et al., 2000). An ideal buffer should have: (i) pH between 6 and 8, preferably 7; (ii) maximum water solubility and minimum solubility in all other solvents; (iii) minimum salt effects; (iv) minimum buffer concentration; (v) the least temperature effect; (vi) good cation interactions; (vii) greater ionic strengths; and (viii) chemical stability (Andrabi, 2009). Development of a suitable buffering system for cryopreservation of buffalo spermatozoa has been in progress for some time, but in a hit-and-miss empirical fashion. Several studies have concentrated on the use of chemically defined buffers for buffalo semen that were originally evolved for cattle bull semen. For example, citrate or Tris and/or citric acid or Laiciphos (IMV, France; containing Laiciphos in unknown buffer) or Biociphos (IMV, France; containing Biociphos in unknown buffer) or Bioxcell (IMV, France; containing unknown buffer and animal protein-free formulae of non-toxic cryoprotectant) have been tested as buffers for the deep freezing of water buffalo spermatozoa (Chinnaiya and Ganguli, 1980a,b; Matharoo and Singh, 1980; Ahmad et al., 1986; Dhami and Kodagali, 1990; Singh et al., 1990, 1991, 2000; Dhami et al., 1994; Akhter et al., 2010). Tris-based buffer has been reported as the most suitable. Zwitterion buffers such as Tes and Hepes have also been used for deep freezing buffalo spermatozoa, but with varying success (Oba et al., 1994; Chachur et al., 1997; Rasul et al., 2000). A study with Bioxcell indicated that it can be an alternative to the laboratory-made Tris-based extender for the cryopreservation of buffalo semen provided that the fertility results on a large scale are found to be satisfactory (Akhter et al., 2010).
From the results of the aforementioned studies, it is suggested that buffers, particularly Tris-based, may provide the most satisfactory system to improve the post-thaw freezability of buffalo spermatozoa. It is believed that Tris-based buffer has a pH nearer to the pKa
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