Introduction. The quality of frozen semen is the most influenced factor of conception rate in the field (Saacke et al., 1994).

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1 Introduction Buffalo population is worldwide distributed and estimated to be over 160 million (FAO, 2003). More than 90% of the population is in Asia where buffaloes play a prominent socioeconomic role in rural livestock production. In recent decades, buffalo farming has expanded widely in Mediterranean areas and in Latin America, but also in central / northern Europe where several herds were introduced. In Egypt, the estimated buffalo population was found to be millions (FAO, 2005) producing about 0.270; 3,752 and 2,300 million tons of meat, hides and milk. The application of AI in animal breeding strategies has been shown to have the potential to quickly disseminate genes from the supergenetic males for improving productive traits. In buffaloes such approach has always been marginal because of low conception rate following both natural and AI (Zicarelli et al., 1997). The causes of reduced fertility in buffaloes have long been considered multifactorial and range from inadequate female management to poor intrinsic semen quality. The male factor remains a common cause of infertility; thus, accurate in vitro assessment of semen quality and improvement of buffalo bull semen cryopreservation are of most important in buffalo breeding. The quality of frozen semen is the most influenced factor of conception rate in the field (Saacke et al., 1994). 1

2 Improvement of buffalo bull semen cryopreservation requires a better understanding of the properties of the currently used extenders. The first aim of sperm freezing protocols is to prevent lethal ice crystal formation and to reduce membrane damage during and after cryopreservation. The discovery of cryoprotective properties of glycerol in 1949 (Polge et al., 1949) led to the ability of spermatozoa for freezing. To be useful for artificial insemination, diluted semen should have a minimum self-life between 2 and 4 days for easy transport and use in distant locations. The predominant effect of storage at 5ºC was to lower the metabolic rate of spermatozoa to be extended survival. The stresses associated with freezing are primarily the temperature changes that spermatozoa is exposed during the process of cooling, the injurious effects of the media components, cryoprotectants themselves during the process and finally the effects of thawing (Watson, 1995). The composition of the extender in which the semen is diluted before freezing is one of the main factors that influence the success of cryopreservation (De Leeuw et al., 1993; Dhami et al., 1994; Woelders et al., 1997). Although several potential cryoprotective substances have been examined e.g. Dimethyl Sulfoxide (DMSO) and propandiol, glycerol has remained the cryoprotectant of choice for spermatozoa from all species. 2

3 In addition to the cryoprotectant, glycerol, the basic components of diluents for freezing of semen that maintain osmolarity and provide buffering capacity; a source of lipoprotein or high molecular weight material to prevent cold shock such as egg yolk, milk or soya lecithin; glucose or fructose as an energy source and other additives, such as enzymes and antibiotics (Vishwanath and Shannon, 2000). Egg yolk-milk extenders are known to provide excellent protections for bull semen against cold shock. However, in recent years there has been increasing arguments against the presence of egg yolk and milk due to the extremely wide variability of their composition based on their initial source and to the constant risk of contamination by bacteria and mycoplasma. Today, a new generation of semen extenders free of animal origin based on the presence of commercially available soybean extract as alternative to egg yolk (Bioxcell, IMV, France). Because of the presence of substances in egg yolk that inhibit the sperm respiration or reduce its motility, there is an increase of the demand to replace the whole egg yolk by the cryoprotective fraction, low density lipoprotein, LDL (Moussa et al., 2002 and Lamia-Amirat et al., 2004). Disaccharides seem to be effective in stabilizing biomembrane bilayers and the sperm metabolism can be better sustained in diluents containing degradable sugar (Aisen et al., 2005). Addition of sucrose and trehalose for freezing of bull semen resulted in an improvement of the sperm survival (Crowe and Clegg, 1987; Westh and Ramlov, 1991; De 3

4 Leeuw et al., 1993 and Woelder et al., 1997). Nonpermeable sugars (lactose, sucrose, trehalose and raffinose) render hypertonic media decreasing the intercellular freezable water and then the amount of cell injury caused by ice crystallization (Schmeil et al., 1986). Trehalose has a protective action related to the osmotic effect and to specific interactions with membrane phospholipids replacing water at the membrane solution interface (Bakas and Disalvo, 1991). The present study was designed to : 1-Identify the effect of different concentrations of LDL on buffalo semen freezability in comparison with Tris-Egg-Yolk fructose. 2-Identify the effect of disaccharides (trehalose and sucrose) with different glycerol concentrations on buffalo semen freezability in comparison with Tris-Egg-Yolk fructose, and validate the optimum concentrations to obtain and improve freezability of buffalo semen. 3-Display the effect of glycerol with different disaccharides (trehalose and sucrose) concentrations on buffalo semen freezability in comparison with Tris-Egg-Yolk fructose, and validate the optimum concentrations to obtain and improve freezability of buffalo semen. 4-Assess the effect of some commercially available extenders containing egg-yolk (Triladyl ) or milk (Laiciphos ) or soybean-based (Bioxcell, IMV, France) extender on sperm characteristics of frozen buffalo bull semen. 4

5 Review of Literature 1. Effect of LDL on freezability of buffalo semen: Hen s egg yolk (EY) is an integral part of the extenders used for bovine semen. The low-density lipoprotein fraction (LDL) of EY appears to be responsible for protection of sperm against freezing damages (Moussa et al., 2002). Many of the hypotheses put forward for mechanisms of sperm protection by LDL suggested an association of LDL with sperm membranes (Cookson et al., 1984). EY-LDL prevents the loss of membrane phospholipids (Parks and Graham, 1992; Bergeron et al., 2004). Though, the precise role of most components of semen extenders have been known, the mechanism of protection provided by EY seems to be much complex and desires further studies. LDL can be safely replaced egg yolk in Tris extender used for freezing buffalo semen with better results in term of sperm motility, membrane integrity, acrosome integrity, live sperm % and total sperm abnormalities % as well as better protection against sperm DNA fragmentation (El-Sheshtawy et al., 2009). Previous experiments proved that LDL possesses remarkable cryoprotective properties for freezing semen at concentration of 8% for bull (Moussa et al., 2002 and Lamia Amirat et al., 2004) and male goat (Ali Al Ahmad et al., 2008), 9% for boar (Jiang et al., 2007) and 6% for canine (Bencharif et al., 2008). The increase of LDL concentration above 10% was shown to result in a decrease in bull spermatozoa performance 5

6 after freeze-thaw presumably due the precipitation of fructose and salts contained in the extender (Moussa et al., 2002). LDL is composed of 85 90% lipids and 10 15% proteins, which is based on a triglyceride core surrounded by a film of proteins and phospholipids and is responsible for the cryoprotective effect of egg yolk during freeze thaw processing of mammalian spermatozoa. The phospholipids that elaborated from disruption during freezing thawing could form a protective film at the surface of the sperm membranes (Hu et al., 2006). LDL is also responsible for the gelation process in freezing and thawing (Moussa et al., 2002). The gelation is the disruption of the LDL structure and this disruption is favored by the dehydration that is caused by the freeze thaw process. On the other hand, some components in the whole egg yolk play an antagonistic role to the cryoprotective effects of LDL (Pace and Graham, 1994). Demianowicz and Strzezek (1996) separated yolk into two lipoproteins: low density lipoprotein (LDL) and high density lipoprotein (HDL) and hypothesized that HDL in yolk have a detrimental effect on its cryoprotective properties. Bergeron et al., (2004) proposed that the principal mechanism by which LDL protects the spermatozoa is via the sequestration of BSP proteins in seminal plasma, considering that the major proteins of bull seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3 and BSP-30-kDa) bind to the sperm surface at ejaculation and stimulate cholesterol and phospholipids efflux from the sperm membrane. 6

7 Since LDL interact specifically with BSP proteins (Bergeron et al., 2004), this would decrease the binding of the major proteins of bovine seminal plasma to the sperm and would prevent lipid efflux from the sperm membrane, which could explain its beneficial effect. 2. Effect of glycerol on freezability of buffalo semen: The discovery of the cryoprotective properties of glycerol in 1949 (Polge et al., 1949) led to ability of spermatozoa for freezing. Although several potential cryoprotective substances have been examined (e.g. DMSO and propanediol), glycerol has remained the cryoprotectant of choice for spermatozoa from all species. The preferential cryoprotective action of glycerol may be attributable to its ability to penetrate sperm membrane more readily than the high molecular weight disaccharides. The main cause of cell damage is the formation of intra- and extacellular ice crystals during freezing. Glycerol and disaccharides are believed to protect the cells by minimizing ice crystal formation. It is known that glycerol is a permeating cryoprotectant able to cross the cell membrane and results in a lower salt concentration of the unfrozen fraction, less shrinking of the cells and larger volume of the unfrozen fraction (Mazur and Rigopoulos, 1983) but the basis of its cryoprotective properties is still unclear. Anchordoguy et al., (1987) noticed that glycerol has a strong affinity with the cells phospholipid head groups during freezing protecting it from cryoinjury. 7

8 Woelders et al., (1997) reported that glycerol modifies the crystal formation in the medium by preventing the electrolyte concentration from rising above a harmful level. Researchers have found significant effects on the motility of bull sperm frozen in buffered egg-yolk and milk extenders in ampules or straws, with varying concentrations of glycerol, different equilibration times and cooling conditions (Parkinson and Whitfieldet, 1987 ; Chen et al., 1993b), but up to 50% of the sperms have been killed by the methods used. Conventional methods of bull semen freezing in or 0.5-ml straws have used approximately 7% glycerol as the optimum concentration for citrate yolk and Tris egg yolk extenders (Watson, 1990 and De Leeuw et al., 1993). Bonia et al., (1980) reported that optimum glycerol levels for egg yolk citrate, egg yolk lactose and egg yolk tris glucose diluents were 7, 9 and 6% respectively. Jainuden and Dass (1982) noted that the percentage of motile spermatozoa was greater in 5% glycerol extender ( ) than in 3 and 7 % glycerol extender ( and respectively), whereas the percentage of intact acrosomes was greater in spermatozoa extended in 3 or 5% glycerol ( and , respectively) than spermatozoa extended in 7% glycerol ( ). Foote (1984) reported a concentration of 6.4% glycerol to be optimal for bull semen. 8

9 Tuli et al., (1985) noticed that buffalo semen diluted in Tris with 7% glycerol and frozen in 0.5 ml straws then thawed at 20 C/2 min, 35 C/1 min, 50 C/30 sec., gave motility averaged 21.85%, %, 36.15% respectively. Fahy (1986) recorded the glycerol concentration used for freezing of bovine semen to be 0.25M (2.25%) and 1M (9%). Fiser and Fairfull (1986) indicated that glycerol provides cryopreservation partly on a basis by reducing the amount of ice formation and partly kinetically by increasing the time for water to leave the cell in response to the decreased vapor pressure of adjacent ice. Garcia and Graham (1987) noticed that freezing of bovine semen with 6% glycerol extender resulted in higher sperm motility than freezing in 3% glycerol. Ismail et al., (1990) found that lactose diluent with 2-3% glycerol results in better sperm revival for pellet freezing of buffalo semen. Fiser et al., (1990) proved that lower glycerol concentrations are required for sperm that are difficult to freeze, such as boar semen, so optimal glycerol concentrations cannot be extrapolated across species or within species from one semen extender to another. Chohan et al., (1992) found that glycerol is the most popular cryoprotectant for buffalo semen cryopreservation. Kumar et al., (1992) noticed that glycerol is commonly added to the extender in concentrations of 6 or 7% for freezing buffalo semen. 9

10 Ziada et al., (1992) noticed that the reduction in post-thawing motility of buffalo semen frozen in Tris with 6.4 % glycerol, Tris with 10 % glycerol, and skim milk with 7% glycerol was much less than in other diluents tested (skim milk with 4 or 7 % glycerol, Tris with 4 % glycerol or lactose-citrate-phosphate with 4, 7 or 10 % glycerol ). De Leeuw et al., (1993) found no significant influence of glycerol concentration between 0 and 8% (v/v) on post-thaw membrane integrity of bull sperm. Gao et al., (1995) reported that the cryoprotection of glycerol is due to its ability to buffer the salt at low temperature, bind with metallic ions, dehydrate the cell, and reduce the ice expansion during water solidification. Woelders and Malva (1998) found a small influence of glycerol, with better survival at 7.3 % compared to 3.68 % glycerol. Chaveiro et al., (2006) found a significant effect of glycerol concentration, amounting to approximately 10 percentage points higher live and motile sperm percentage at 800mM, compared to 400mM glycerol. Rasul et al., (2007) studied the effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm from Nili-Ravi buffalo. They found that glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), and plasma membrane integrity. The addition of glycerol (6%) at 37 C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 C. 10

11 3. Effect of disaccharides on freezability of buffalo semen: Previous efforts to improve methods for cryopreservation of bull semen include the use of trehalose and sucrose. It has been demonstrated with artificial membranes that damage measured by intermixing and fusion could be reduced by a series of cryoprotectant, with trehalose and sucrose being more protective than glycerol (Rudolph et al., 1986 and Anchordoguy et al., 1987). Platov (1988) reported that disaccharides are more effective than monosaccharides for increasing the osmotic dehydration. This osmotic effect decreases the intracellular freezable water, and then the amount of cell injury by ice crystallization. Garcia and Graham (1989) pointed out that disaccharides or monosaccharides are effective than trisaccharides for the preservation of post-thaw motility of bovine spermatozoa. Disaccharides were considered as non permanent sugars because they do not penetrate the plasma membrane of the sperm cell rendering hypertonic media cause sperm cells to loose water and shrink before freezing (Aisen et al., 1990). Although, sugars are routinely included in the composition of many extenders, its protective action on spermatozoa is still little understood (Maxwell and Salamon, 1993). The presence of these sugars in the diluents is likely to affect the pattern of ice crystallization and the shape and width of the channels of the unfrozen solution which may relieve or prevent fast-cooling damage of spermatozoa (Nicolajsen and Hvidt, 1994). 11

12 Many authors have shown that sugars protect against the damage occurring to sperm cells during freezing-thawing depending on many factors, such as storage temperature (Lapwood and Martin, 1966), the type of buffer used (Abdelhakeam et al., 1991) and the molecular weight of sugar (Molinia et al., 1994a). Molinia et al., (1994b) noticed that the motility of frozen- thawed ram spermatozoa is higher in the presence of sucrose or trehalose than in the presence of glucose when glycerol is not incorporated in the diluent; whereas when glycerol is employed in the diluent, no differences were observed among the various sugar types. Moreover, disaccharides are reported to be effective in stabilizing sperm membrane bilayers by forming hydrogen bonds with the polar head groups of the phospholipids thus substituting for the water molecules under dehydration (De-Leeuw et al., 1993 and Woelders et al., 1997). Sperm metabolism can be better sustained in diluents containing degradable sugars although iso-osmotic diluents are commonly used as semen extenders, hyperosmotic diluents over in a wide range of sugars concentrations, are known to improve sperm integrity after freezingthawing (Vekseler et al., 1997). Woelders et al., (1997) demonstrated that an isotonic sugar medium in which Tris citrate components were substituted with sucrose and trehalose is significantly superior to a Tris-citrate egg yolk medium in preserving the motility and acrosome integrity of bovine spermatozoa. Woelders and Malva (1998) shown a significant interaction between disaccharides and glycerol concentration but this interaction was quite modest. 12

13 Addition of sugars would lead to a lower salt concentration in the unfrozen water, consequently reducing the injury due to the solution effects. It is also possible that sugars may help to prevent injurious eutectic freezing by trapping salts in an increasingly viscous or even glasslike phase (Wakayama et al., 1998). Furthermore, these sugars could interact directly with membrane lipids and proteins, inducing a depression of the membrane phase transition temperature of dry lipids, forming a glass (vitrification) during dehydration and rehydration along cryopreservation (Aboagla and Terada, 2003 and Hincha et al., 2006) Effect of trehalose on freezability of buffalo semen: Several investigators have found that the incorporation of trehalose in sperm diluents protect the spermatozoa of many species against freeze damage. Trehalose (α, α-trehalose) is a disaccharide formed by a 1,1 linkage of two d-glucose molecules. It is a non-reducing sugar that is not easily hydrolyzed by acid, and the glycoside bond is not cleaved by glycosidase. The molecular formula and weight are C 12 H 22 O 11 and , respectively. When purified, it is usually found in the dihydrate form, which is the typical commercial product. Physical properties that make trehalose unique are its high degree of optical rotation ([a] D ) and its melting behavior. Trehalose first melts at 97 C. Additional heat drives off the water of crystallization until the material resolidifies at 130 C, and then the anhydrous trehalose melts at 203 C. The combination of the molecular 13

14 structure and physicochemical properties of trehalose result in a very stable disaccharide (Elbein, 1974). Although α, ß (neotrehalose) and ß, ß (isotrehalose) isomers of trehalose have been synthesized, they are rarely found in nature. The α, α form is the common isomer (α, α -trehalose, α -D-glucopyranosyl α -Dglucopyranoside, mushroom sugar, mycose), and is the widespread. Trehalose is found in a number of plants and animals that can resist dehydration or freezing (Crowe et al., 1987). It can form hydrogen bonds with the polar head of groups of phospholipids and thus helps prevent fusion events of juxtaposed membranes and with respect to, trehalose supplemented in extenders of various species semen cryopreservation. Although, many authors supposed the mechanism of trehalose to protect sperm during freezing/thawing process as mentioned before but the precise mechanism remains to be elucidated and the exact mechanism of fast-cooling damage remains a subject of debate (Westh et al., 1991). In addition to its dehydrating effect, trehalose confers a specific cryoprotection on the lipid bilayer (Bakas et al., 1991). Van Kalsbeek (1992) performed some experiments in the Dutch and the German cattle AI organization testing the effect of trehalose in a concentration of 15 g/100 ml of standard freezing medium, which makes the medium distinctly hypertonic. Although the motility of the sperm was inhibited in this hypertonic trehalose medium, the percentage of sperms with intact membrane-integrity after freezing and thawing was higher in the trehalose medium than in isotonic standard Tris egg yolk medium. 14

15 Chen et al., (1993a) also investigated the effect of trehalose in freezing medium for bull semen on the viability of the sperm after freezing and thawing and reported no positive contribution of trehalose. It is conceivable that the ability of trehalose to insert itself into bilayer phospholipids could modulate membrane fluidity, rendering the sperm membrane more able to withstand damage from freezing (Iwashi et al., 1995). Liu et al., (1998 c), in a study on the freezability of bull spermatozoa in Tris-Citrate-Glycerol (TCG) extender containing up to 25% (v/v) trehalose or sucrose, concluded that replacing part of TCG-containing egg yolk with these sugars had no beneficial effects. Storey et al., (1998) showed that trehalose brings about a significantly better recovery rate in intact mouse spermatozoa when compared with raffinose. Yildiz et al., (2000) examined the influence of different sugars supplemented to the Tris-egg yolk extender on the motility of frozen thawed dog sperm. They showed that when sperm were frozen with trehalose added to Tris-egg yolk extender; this improved the motility of frozen thawed sperm as compared with other sugars, such as glucose, lactose and sucrose. Aisen et al., (2000) observed that trehalose significantly improves the viability of ram spermatozoa assessed for motility and acrosome integrity, with the best results obtained for a trehalose + EDTA extender. Aisen et al., (2002) reported that the trehalose-mediated cryoprotection is increased by glycerol, i.e., presenting a synergistic effect. 15

16 Aisen et al., (2002) indicated that addition of 100mM trehalose to the extender improved ram sperm motility after thawing by 35% with respect to the control diluent without trehalose and gave 45 47% lambing by cervical insemination in two consecutive trials with 2.5 times greater than the fertility rate obtained with control diluent. Nagy et al., (2003) indicated in their research regarding the frozen/thawed bull semen that highest concentrations of trehalose had unfavorable influences on semen characteristics than lower concentrations. Aboagla and Terada (2004) found that higher trehalose concentrations significantly protected goat spermatozoa against freezing damage. Yamashiro et al., (2006) shown that trehalose provide positive protection for freezing of goat semen. Mustafa et al., (2007) found that trehalose at a level of 50mM provided the best maintenance of motility; the highest percentage of viable sperm and the highest percentage of membrane-intact sperm after storage of ram semen at 5 C. Hu et al., (2009) noticed that addition of trehalose up to 100 mm, confers a greater cryoprotective capacity to the extender used for cooling of boar semen at 5 C, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process. While freeze-thawing tolerance diminished significantly for 200mM. Kozdrowski (2009) reported that addition of 100mM trehalose to the extender of brown hare semen result in more significant decrease in a percentage of motile, viable and acrosome intact spermatozoa after cooling 16

17 at 5 C than the addition of 50mM, which was clearly seen after 3 h of incubation. Shiva Shankar Reddy et al., (2010) proved that addition of 50mM taurine or 100mM trehalose to the freezing extender of buffalo semen has shown better post-thaw motility, viability and membrane integrity than extender without these additives Effect of sucrose on freezability of buffalo semen: Sucrose, ordinary table sugar, is probably the single most abundant pure organic chemical in the world and the most widely known to non chemists. Whether from sugar cane (20% by weight) or sugar beets (15% by weight), and whether raw or refined, common sugar is still sucrose. Sucrose is a disaccharide that yields 1eq of glucose and 1eq of fructose on acidic hydrolysis. This 1:1 mixture of glucose and fructose is often referred to as invert sugar, since the sign of optical rotation changes (inverts) during the hydrolysis from sucrose to a glucose fructose mixture. Unlike most other disaccharides, sucrose is not a reducing sugar and does not exhibit mutarotation. These facts imply that sucrose has no hemiacetal linkages and that glucose and fructose must both be glycosides. This can happen only if the two sugars are joined by a glycoside link between C1 of glucose and C2 of fructose. Sucrose like trehalose can stabilize membranes by forming hydrogen bonds with phosphate groups' or membrane phospholipids and thereby may provide additional protection to sperm during swelling and shrinking, processes that sperm go through during freezing and thawing (Crowe et al., 1990). 17

18 De Leeuw et al., (1993) have investigated the effect of addition of sucrose and trehalose to freezing medium for bull semen in a number of pilot experiments. There was a small positive effect on sperm survival with sucrose than with trehalose. Furthermore, sucrose added to EYT was effective in maintaining the integrity of bull sperm membranes during freezing; this proved that substances which stabilize membranes are more beneficial to cryopreserved sperm than are the membrane-penetrating cryoprotectants. Woelders et al., (2002) suggested that sucrose-based freezing media were beneficial for the post-thaw survival of bull sperm both under optimal and suboptimal cooling rates. Moreover, the percentage of live cells after freezing in sucrose medium was a few percentage points higher compared to Tris medium. Chaveiro et al., (2006) found that motility in Tris medium was always better than in sucrose medium. Although, the percentage of live cells after freezing in sucrose medium was a few percentage points higher compared to Tris medium, the sucrose-based medium does not offer a clear consistent benefit compared to the Tris medium. Farshad and Akhondzadeh, (2008) revealed that although sucrose plus glycerol proved better with regard to intensity of motility, progressive motility and live sperm of goat semen, the diluents without glycerol give the best results concerning release of hyaluronidase. Hyaluronidase release into seminal plasma from the acrosome is an early and sensitive indicator of acrosome damage during processing and freezing (Foulkes and Watson, 1995 and Sirat et al., 1996). 18

19 4. Effect of some commercially available extenders on freezability of buffalo semen: Many extenders have been produced, usually on an empirical basis, to protect and maintain the fertilizing ability of spermatozoa during processing and storage of the semen. The most common constituent in semen extenders has historically been the egg's yolk (Foote, 1984). Both egg yolk and glycerol have been used as cryoprotectants for more than 50 years, with a complementary and synergistic action on plasma membrane. In attempting to improve quality standard, the artificial insemination industry has raised concerns regarding the use of such additives, which are diverse and often vary in their composition, hence the quality guarantee become very difficult or often impossible to perform. In addition, they represent a potential risk either via microbial contamination of the extender or otherwise (Bousseau et al., 1998). The use of chemically defined extenders with additives of vegetable origin would be preferable than those of animal origin from a sanitary point of view. At present, an extender containing soybean-derived components as a substitute for egg yolk are commercially available (Biociphos-Plus and Bioxcell ), representing an alternative to the hygienic concerns raised by customers as well as the possibility to simplify the semen handling procedures for the AI industry ( Bousseau and Brillad, 1994 and Gil et al., 2000). A huge number of extenders were examined to choose the best ones that would maintain the fertility of buffalo spermatozoa. 19

20 Chinnaiya and Ganguli (1980b) reported that buffalo semen frozen in Tris diluent had the highest post-thawing motility between other diluents. Nour (1980) reported in a comparison between Tris -citric acid - fructose-yolk (TCFY), egg yolk citrate (EYC), egg yolk- glucosebicarbonate (EYGB), egg yolk- skim milk (EYSM) and Laiciphos-271 that EYSM and Laiciphos yielded higher post-thaw motility than other diluents. Matharoo and Singh (1980) found that the highest post-thawing motility of buffalo spermatozoa was found for semen frozen in Tris-egg yolk glycerol diluent (47.78 %). Ala-Ud-Din et al., (1981) denoted that post-thawing motility and survival time at 37 C did not differ significantly for buffalo semen frozen in homogenized whole milk-egg yolk-glycerol, laiciphos, lactose-egg yolkglycerol, and glucose-egg yolk-citrate glycerol, while the absolute survival was greatest for semen frozen in lactose-egg yolk-glycerol. Tamyurek (1982) used milk glycerol (MG), MG egg yolk and MG fructose for dilution and preservation for bull semen. He reported that sperm motility in fresh semen was 68%, while motility of semen frozen in the three above-mentioned diluents was 56, 61.6 and 54.8% respectively. Upon freezing of Friesian bull semen in five diluents ( lactose-egg yolk-glycerol, fructose-egg yolk, glycine-egg yolk-glycerol, lactosefructose-glycine-egg yolk-glycerol and Laiciphos-glycerol ), Ala-Ud-Din et al., (1985) found that the highest post-thawing motility (50.8 %) and sperm survival rate at 4 C (41.02 %) were obtained with the lactosefructose-glycine-egg yolk-glycerol and the lowest motility (39.67 %) and survival (26.78 %) with lactose-egg yolk-glycerol. 20

21 Dhami et al., (1987) found that the post-thawing motility of buffalo semen frozen in Tris-fructose-egg yolk, egg yolk-citrate and lactose-egg yolk averaged 44.8, 45.6 and 44.3% respectively. Abdel-Rahman (1988) reported that sperm recovery in skim milk diluents was better than in Triladyl. According to Abdel-Malak et al., (1989), post-thawing motility in Tris buffer, has been better at ph than at 6.5 or 8.0, but Tris molarities between 0.20 and 0.35 did not vary significantly. Hazarika et al., (1989) found that egg yolk-tris-cysteine hydrochloride diluent maintained better post-thawing sperm motility of Murrah buffalo semen than other diluents. Ziada et al., (1995a, b) found that commercial diluents Triladyl and Laiciphos resulted in comparable post-thawing motility. Hinsch et al., (1997) reported no significant differences in motility, viability, and acrosome status of spermatozoa between Biociphos!Plus and Triladyl, in spite of higher post thaw proportion of linear motility found for Biociphos!Plus compared to Triladyl extended semen. Hurtado (1998) reported low post thaw linear motility for Biociphos!Plus compared with Triladyl, despite the higher velocity patterns for Biociphos!Plus. Moreover, better membrane integrity in spermatozoa processed in Triladyl than Biociphos Plus. Müller-Schlösser et al., (2001) proved that semen extenders that are free of animal products when compared to conventional ones, no differences have been found in post-thaw-total motilities. 21

22 Herold et al., (2003) indicated that Triladyl secures post-thaw motility as high as fresh semen of African buffalo. Moreover, it gives superior numbers of intact acrosomes. De Pauw et al., (2003) compared the storage capacity of CEP-2 (Cauda Epididymal Plasma CEP) and Tris with Triladyl, and observed that spermatozoa stored in CEP-2 diluent moved significantly faster (velocity straight line VSL, beat cross frequency BCF) and straighter (straightness STR, linearity LIN) than in Triladyl and Caprogen diluent. When sperm motility was evaluated subjectively, increased percentages of hyperactivated spermatozoa were observed in Caprogen and Triladyl diluents. According to Patil et al., (1981) the proportion of abnormal spermatozoa was found to be lower after thawing of frozen buffalo semen in Triladyl and Tris diluents ( ) than in citric acid whey diluent ( ). Kumar et al., (1988) denoted that the percentage of abnormal spermatozoa of buffalo semen frozen in egg yolk-citrate, egg yolk-citrate lecithin, egg yolk-tris and egg yolk Tris-lecithin, averaged 39.33±2.19, 34.58±1.56, 37.16±1.21 and 32.82±2.38, respectively. Chinnaiya and Ganguli (1980a) found that the percentage of spermatozoa with intact acrosomes averaged 55.0, 53.0 and for semen processed in egg yolk citrate, citric acid whey and Tris diluents, respectively. 22

23 Kakar and Anand (1984) presented data showing significant damage to the acrosome of buffalo spermatozoa varying between 37 and 42 % upon freezing in egg yolk-citrate-glucose, egg yolk-tris, skim milk-egg yolk or citric acid-whey. Shannon and Curson (1984) tested the storage capacity of CEP-2 diluent (Cauda Epididymal Plasma CEP) and Tris in comparison with caprogen and no significant differences in membrane and acrosome integrity or in mitochondrial membrane potential were observed between the three diluents during the 6 days of storage. Chandler et al., (1984) noted that freezing of bull semen in egg yolk citrate glycerol, egg yolk-tris-glycerol and whole milk glycerol resulted in improvement of acrosome integrity with increasing final semen temperature after thawing up to 31 C but not to 44 C for any of these diluents. Roy and Choudhry (1987) indicated that there were no significant differences between bull ejaculates diluted with yolk-glucose-citrate diluent containing fresh-egg yolk, a diluent containing freeze-dried egg yolk, kept in a refrigerator or kept at room temperature in the percentages of spermatozoa with normal acrosomes. Ziada (1989) found that the acrosomal maintainance was better for buffalo semen frozen in Triladyl or skim milk containing 4 or 10% glycerol compared to other diluents (Tris, lactose-citrate-phosphate or Laiciphos). 23

24 Singh et al., (1991) found that Tris-egg yolk-glycerol diluent provided the highest protection against acrosome damage of frozen buffalo spermatozoa, followed by egg yolk-citrate-glycerol and citric acid-wheyglycerol. Gil et al., (2000) proved that the clarified milk based extenders provided more effective preservation of the acrosome membrane and viability after thawing compared to TRIS-citrate-fructose extender in ram semen. Gil et al., (2003) obtained better fertility values in extenders containing soybeans than in egg yolk-supplemented extenders as Triladyl or standard Tris used for bull semen. Paulenz et al., (2004) obtained good fertility results with milk based extenders (laiciphos) with antibiotics included for goat semen. Waterhouse et al., (2010) showed an increase in DNA damage after dilution, cooling (5 C) and after freezing thawing of Norwegian Red (NRF) bull semen with skimmed milk egg yolk (SMEY), but only after freezing thawing with Triladyl concluding that Triladyl maintain DNA integrity better than SMEY during cryopreservation. 24