Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa

bảo quản lạnh tinh trùng: Storage of buffalo (Bubalus bubalis) bull semen in the cryopreserved state is discussed in this article. Fertility rate in buffalo following artificial insemination with frozen–thawed semen is reviewed. To better understand the freezability of bubaline spermatozoa, the available data on biochemical components and the activity of specific enzymes of semen⁄spermatozoa are given. Moreover, the major factors that may influence the postthaw viability and fertility of buffalo spermatozoa are examined in detail. In addition, suggestions for improvement in cryogenic procedures for buffalo spermatozoa are also given.

Reprod Dom Anim 44, 552–569 (2009); doi: 10.1111/j.1439-0531.2008.01240.x ISSN 0936-6768 Review Article Factors Affecting the Quality of Cryopreserved Buffalo (Bubalus bubalis) Bull Spermatozoa SMH Andrabi Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan Contents Storage of buffalo (Bubalus bubalis) bull semen in the cryopreserved state is discussed in this article Fertility rate in buffalo following artificial insemination with frozen–thawed semen is reviewed To better understand the freezability of bubaline spermatozoa, the available data on biochemical components and the activity of specific enzymes of semen ⁄ spermatozoa are given Moreover, the major factors that may influence the post-thaw viability and fertility of buffalo spermatozoa are examined in detail In addition, suggestions for improvement in cryogenic procedures for buffalo spermatozoa are also given Introduction The domestic buffalo, Bubalus bubalis, is a distinct species within the Bovidae family The buffalo population is continuously increasing, and is estimated at over 170 million head (Food and Agricultural Organization (FAO) 2004) More than 95% of the population is located in Asia, where buffaloes play a prominent role in rural livestock production providing the milk, meat and work draft force In recent decades, buffalo farming has also expanded widely in Mediterranean areas and in Latin America Only in India and Pakistan are there well-defined buffalo breeds (Drost 2007) There are 18 river buffalo breeds in South Asia, which are further classified into five major groups designated as 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 breeds of buffalo (Cockrill 1974) The swamp buffalo found in Southeast and Far East Asia has low milk production, and is mostly used as a draft animal by small farm holder or is utilized for meat purpose The production potential of livestock can be increased by genetic improvement using one of the modern ways of breed improvement, e.g., artificial insemination (AI) Moreover, the quality of frozen–thawed semen is one of the most influential factors affecting the likelihood of conception (Saacke 1984) Application of AI with frozen– thawed semen has been reported on a limited scale in buffalo, because of poor freezability and fertility of buffalo spermatozoa when compared with cattle spermatozoa (Kakar and Anand 1981; Muer et al 1988; Raizada et al 1990; Singh and Pant 2000; Andrabi et al 2001, 2008; Ahmad et al 2003; Senatore et al 2004; Kumaresan et al 2005) Hence, successful cryopreservation of bubaline semen would aid in the creation of long-term storage of male gametes and the maintenance of genetic stock that could improve milk and beef production and its associated economic value internationally This article deals with the storage of bubaline spermatozoa in deep-frozen ()196°C) state and reviews the major factors affecting the viability and fertility of cryopreserved buffalo spermatozoa Cryopreservation of Spermatozoa Cryopreservation is a non-physiological method that involves a high level of adaptation of biological cells to the osmotic and thermic shocks that occur both during the dilution, cooling–freezing and during the thawing procedures (Watson et al 1992; Holt 2000a,b) Damage occurring during the freezing–thawing procedures affect mainly cellular membranes (plasma and mitochondrial) and in the worst case, the nucleus (Blesbois 2007) This damage to membranes has consequences on viability and different metabolic factors including adenosine triphosphate (ATP) concentration in spermatozoa Therefore, such changes in the integrity of spermatozoa affect the viability and fertility Table summarizes different stresses encountered by the cell and the effect on the cell of each stressor during the cryogenic processes The first successful freezing of buffalo semen was reported by Roy et al (1956) Basirov (1964) was the first to report the pregnancy with frozen–thawed buffalo bull spermatozoa Since then, AI has been adopted in buffaloes; however, it remains unpopular because of poor fertility rate with frozen–thawed semen (Muer et al 1988; Andrabi et al 2001; Ahmad et al 2003; Senatore et al 2004; Kumaresan et al 2005, 2006; Shukla and Misra 2007) A summary of available studies on fertility of frozen buffalo spermatozoa with AI is presented in Table A critical assessment in term of first service conception rate of the reports given in Table is difficult, as in most of the studies; the number of inseminations was low Details such as number of spermatozoa per dose of AI and freezing protocol were not provided for some of the studies Few studies even lacked the basic information, like on the type of extender used for cryopreservation or the total number of animals inseminated However, despite the shortcomings in the published reports (Table 2), it can be suggested that the pregnancy rate in buffalo with AI using frozen–thawed semen is not comparable with that of cattle Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa Table Sources of injury from freeze-thawing of cells (Morris and Clarke 1987) Stress encountered Temperature reduction Increased solute concentration Increased ionic concentration Dehydration Precipitation of salts and eutectic formation Gas bubble formation Increased solution viscosity Changes in pH Direct contact between cells Potential cellular response Membrane lipid phase changes and depolymerization of the cytoskeleton Osmotic shrinkage Direct effects on membranes, including solubilization of membrane proteins Destabilization of the lipid bilayers Unknown Mechanical damage to membranes and the cytoskeleton Possible limitation of diffusion processes Denaturation of proteins Membrane damage Conception rate in buffaloes inseminated with frozen– thawed semen under field condition is approximately 30% (Chohan et al 1992; Anzar et al 2003) Published reliable studies on the fertility of liquid stored buffalo semen seem not to be available (Sansone et al 2000) However, few scattered reports indicate a pregnancy rate of approximately 60% with liquid semen AI in buffaloes (Tomar and Singh 1970; Akhter et al 2007) Therefore, a pregnancy rate higher than 50% is regarded as a good result after AI with frozen–thawed spermatozoa in buffalo (Vale 1997) It is relevant to mention that the same pregnancy rates i.e., near 50% under normal circumstances are considered poor in cattle with frozen–thawed spermatozoa From above, it is suggested that cryopreservation adversely affects the viability and the fertilizing potential of buffalo bull spermatozoa Therefore, there is a need to discuss in depth the major factors influencing the successful cryopreservation of buffalo spermatozoa Factors Affecting Freezability Biochemical characteristics of semen It is reported that buffalo spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Raizada et al 1990; Andrabi et al 2008) Moreover, there are specific biochemical factors that affect the ability of spermatozoa to prevent damages caused by the cryogenic procedures One of the many possible causes of lower freezability of buffalo bull semen compared to cattle bull can be due to the differences in the lipid ratio of the spermatozoa (Jain and Anand 1976; Tatham 2000; European Regional Focal Point on Animal Genetic Resources, 2003) For example, phosphatidyl choline makes up approximately 66% of all phospholipids found in buffalo sperm plasma membrane (Cheshmedjieva and Dimov 1994) but approximately 50% in case of cattle bull sperm membrane (Parks et al 1987) Similarly, phosphatidyl ethanolamine makes up approximately 23% of all phospholipids present in buffalo sperm plasmalemma (Cheshmedjieva and Dimov 1994) but almost 10% in case of cattle bull sperm membrane (Parks et al 1987) To better, understand the nature of bubaline spermatozoa the available data on biochemical components and the activity of specific enzymes of semen are given in 553 Tables and The values of different constituents given in Tables and show that buffalo whole semen ⁄ seminal plasma ⁄ spermatozoa ⁄ plasma membrane compared to cattle have distinct characteristics, particularly the membrane lipid ratio Therefore, there is a need to develop biochemically defined extenders and cryogenic procedures that are species specific, and may result in the improvement of viability and fertility of frozen–thawed buffalo spermatozoa Buffer 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 and 8, preferably 7; (ii) maximum water solubility and minimum solubility in all other solvents; (iii) minimum salt effects; (iv) minimum buffer concentration; (v) least temperature effect; (vi) wellbehaved cation interactions; (vii) greater ionic strengths and (viii) chemical stability (Bates 1961; Good et al 1966; Good and Izawa 1972; Keith and Morrison 1981) Development of a suitable buffering system for the cryopreservation of buffalo spermatozoa has been in progress for sometime (Rasul et al 2000) Several studies have concentrated on the use of chemically defined buffers for buffalo semen In this regard, Matharoo and Singh (1980) tested citrate, Tris or citric acid as buffers for deep-freezing of buffalo spermatozoa They found that freezing loss was least with Tris-based extender as judged by post-thaw motility Similarly, Chinnaiya and Ganguli (1980a) found better post-thaw sperm motility with Tris-based extender than citrate or citric acid-based extenders In another study, Chinnaiya and Ganguli (1980b) found that spermatozoon frozen in citric acid, citrate or Tris-based extender showed similar degree of acrosomal damage and similar recovery rates However, acrosin activity was greatest in citrate-based diluent and least in Tris buffer Ahmad et al (1986) found that Tris–citric acid based extender is suitable for the cryopreservation of buffalo spermatozoa in terms of post-thaw motility and survivability Later on, Dhami and Kodagali (1990) studied the effects of semen extenders based on Tris or citrate buffer It was reported that Tris-based extender improved the freezability of buffalo spermatozoa as judged by the extracellular release of spermatozoal enzymes and in vivo fertility Similarly, Singh et al (1990, 1991) studied semen diluents based on citrate or Tris or citric acid for freezing of buffalo spermatozoa They found that with Tris-based extender there was least release of lactic dehydrogenase and sorbitol dehydrogenase in buffalo spermatozoa during cryopreservation followed by citrate and citric acid-based extenders In addition, Tris provided the highest protection against acrosomal damage compared to other buffers tested Dhami et al (1994) studied the effects of semen extenders based on Tris or citrate It was found that Tris-based extender yielded higher post-thaw spermatozoal motility Singh et al (2000) compared Tris-buffer with Laiciphos (IMV, L’Aigle, France; containing laiciphos, egg yolk, distilled water and unknown buffer) and Biociphos (IMV, France; containing biociphos with Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 554 SMH Andrabi Table Fertility rate in buffalo following AI with frozen–thawed semen Reference Bhosrekar and Nagarcenkar 1971 Bandyopadhyay and Roy 1975 Chinnaiya et al 1979 Singh et al 1980 Tuli et al 1981a Matharoo and Garcha 1986 Heuer and Bajwa 1986 Heuer et al 1987 Singh and Singh 1988 Ahmad et al 1988 Bhavsar et al 1988 Bhavsar et al 1989a Bhavsar et al 1989b Singh 1990 Haranath et al 1990 Dhami and Kodagali 1990 Dhami and Kodagali 1991 Hassan and Zia Ur 1994 Dhami et al 1994 Barnabe et al 1994 Dhami et al 1996 Younis et al 1999 Barile et al 1999b Gokhale and Bhagat 2000 Sukhato et al 2001c Pant et al 2001 Prabhakar et al 2002 Taraphder et al 2003 Sosa et al 2003 Presicce et al 2004d Kanchan and Singh 2005 Anzar et al 2003 Andrabi et al 2006 Extender Total number of first AI Skim milk powder–yolk–glycine–citrate– fructose–glycerol Yolk–citrate–glycerol Yolk–citrate–glycerol, Citric acid–whey–glycerol and Tris–yolk–glycerol Tris–yolk–glycerol Tris–yolk–glycerol Tris–yolk–glycerol Information not provided Lactose–fructose–yolk–glycerol, Skim milk–fructose–yolk–glycerol and Tris–fructose–yolk–glycerol Information not available Yolk–glycerol with milk or lactose or fructose and lactose Yolk–lactose–fructose–glycerol Tris–fructose–yolk–glycerol Tris–fructose–yolk–glycerol with or without additives (L-cysteine HCl H2O, sheep hyaluronidase, beta-amylase or acetylcholine chloride) Information not provided Tris–egg yolk–glycerol Tris–fructose–yolk–glycerol, Yolk–citrate–glycerol and Lactose–yolk glycerol Information not provided Information not provided Tris–yolk–glycerol–, Citrate–yolk–glycerol– and Lactose–yolk–glycerol–, with or without (control) cysteine, EDTA and raffinose Tris–TES and Tris–yolk Tris–citric acid–fructose–yolk–glycerol and Whole cow’s milk–yolk–glycerol Lactose–fructose–yolk–glycerol Information not available Information not provided Tris–fructose–yolk–glycerol Information not provided Information not available Information not provided Milk-, Laiciphos- and Tris- with or without glycerol, DMSO and propylene glycol Information not available Information not available Information not available Tris–citric acid–fructose–yolk–glycerol Over all first service aCR (%) 109 45.0 Information not available 315 40.6 53.93 72 159 825 61 952 3220 45.8 35.22 39.30 51.78 37.4 218 2745 41.0 44.7 1908 Information not available 3791 39.2 45.85 46.0 Information not provided Information not available 3412 39.7 51.53 39.9 2995 1110 853 40.1 65.26 57.95 Information not available 806 53.14 63.98 971 217 6762 178 202 1941 Information not available Information not available 41.8 42.5 52.0 37.0 34.95 59.15 40.75 50.6 67 Information not available Information not available 432 48.0 29.87 29.0 53.0 a Pregnancies were confirmed through rectal palpations Buffalo were synchronized with a progesterone-releasing intravagiral device (PRID) containing progesterone and oestradiol benzoate, for 10 days Seven days after insertion of PRID the buffalo received an injection of pregnant mare serum gonadotropin (PMSG) and an injection of cloprostenol AI was performed 48, 72 or 96 h after removal of the device c Oestrus synchronization was performed by inserting a progesterone-impregnated silicone elastomer device (CIDR-BÒ) into the vagina Each buffalo was injected intramuscularly with mg of oestradiol benzoate (CIDIROLÒ) on the day of CIDR-B insertion and 150 IU of ECG upon CIDR-B removal (12 days after insertion) AI was performed between 48 and 50 h after the CIDR-B was removed d AI was performed twice at 72 and 96 h after administration of prostaglandins to buffaloes bearing a functional corpus luteum b glycerol, egg yolk, distilled water and unknown buffer) for cryopreservation of buffalo semen Again they found that Tris-based extender was better compared to Laiciphos and Biociphos as judged by post-thaw motility and survivability Rasul et al (2000) carried out a study to identify the suitable buffer for cryopreservation of buffalo semen The buffers tested were tri-sodium citrate, Tris–citric acid, Tris–Tes or Tris–Hepes They found that Tris– citrate tended to be better in term of improving the postthaw motion characteristics of buffalo spermatozoa Nonetheless, plasma membrane integrity and normal acrosomes of spermatozoa did not vary because of buffering systems Conversely, Oba et al (1994) and Chachur et al (1997) found that Tes is to be equal value to Tris-based extender in terms of post-thaw motility, acrosome retention or membrane integrity From the results of the above mentioned studies, it is suggested that zwitterion buffers particularly, Tris–citric acid may provide the most satisfactory buffering system to improve the post-thaw freezability and consequently may also improve the fertility of buffalo spermatozoa It is believed that zwitterion buffers have pH nearer to the pKa (acid dissociation constant) Also there pKa is least influenced by temperature as compared to other buffers (Graham et al 1972) Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa 555 Table Biochemical composition of buffalo semen Characteristic of component Reference Whole semen Seminal plasma Spermatozoa Rattan et al 1980 23.47 mg ⁄ 100 ml Dabas et al 1984 82 ± mg ⁄ 100 ml 167 ± lg ⁄ 1011 cells 0.024 ± 0.003 lmol ⁄ ml 3.9 ± 0.5 mg ⁄ 100 ml 0.066 ± 0.014 lmol ⁄ 109cells Banerjee and Ganguli 1973 0.091 ± 0.011 lmol ⁄ ml 6.2 ± 0.8 mg ⁄ 100 ml Citric acid Banerjee and Ganguli 1973 441.8 ± 31.9 mg ⁄ 100 ml 444.9 ± 17.4 mg ⁄ 100 ml Fructose Salem and Osman 1972 Lactic acid Ascorbic acid Jain 1987 Banerjee and Ganguli 1973 Rattan et al 1980 Lipids 368.12– 430.92 mg ⁄ 100 ml 815.71 mg ⁄ 100 ml 1.500 mg ⁄ ml 1.147 mg ⁄ 109 cells Sarmah et al 1983 1.750 ± 0.030 mg ⁄ ml 1.320 ± 0.030 mg ⁄ 109 cells Cholesterol Mohan et al 1979 Phospholipids Jain and Anand 1976 0.594 mg ⁄ ml 0.548 mg ⁄ 109 cells Sidhu and Guraya 1979 0.1735 ± 0.0256 mg ⁄ ml 0.069 ± 0.02 mg ⁄ ml 0.3074 ± 0.0923 mg ⁄ 109 cells 0.064 ± 0.02 mg ⁄ 109 cells Jain and Anand 1976 21.7 ± 1.0% of total phospholipids 30.4 ± 1.4% of total phospholipids Sarmah et al 1983 34.1 ± 1.8% of total phospholipids 28.0 ± 1.2% of total phospholipids Jain and Anand 1976 17.3 ± 0.9% of total phospholipids 19.4 ± 1.7% of total phospholipids Phosphatidyl choline Phosphatidal choline (choline plasmogen) Amount of lactic acid in cattle bull seminal plasma is 72 ± mg ⁄ 100 ml (Dabas et al 1984) Amount of lactic acid in cattle bull spermatozoa is 352 ± 16 lg ⁄ 1011 cells (Dabas et al 1984) Amount of ascorbic acid in whole semen, seminal plasma and spermatozoa of cattle bull is 0.131 ± 0.030, 0.505 ± 0.0185 lmol ⁄ ml and 0.0832 ± 0.0337 lmol ⁄ 109cells, respectively (Jain 1987) Amount of citric acid in whole semen and seminal plasma of cattle bull is 531.3 ± 73.4 and 576.9 ± 58.6 mg ⁄ 100 ml, respectively (Banerjee and Ganguli 1973) Amount of fructose in seminal plasma of cattle bull is 519.07–618.93 mg ⁄ 100 ml (Salem and Osman 1972) 623.8 ± 83.6 mg ⁄ 100 ml Jain and Anand 1976 Sarmah et al 1983 Comment 91.84 ± 3.91– 141.88 ± 3.12 mg ⁄ 100 ml Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Amount of fructose in whole semen of cattle bull is 780.6 ± 66.2 mg ⁄ 100 ml (Banerjee and Ganguli 1973) Amount of lipids in seminal plasma and spermatozoa of cattle bull is 2.900 mg ⁄ ml and 0.703 mg ⁄ 109 cells, respectively (Jain and Anand 1976) Amount of lipids in seminal plasma and spermatozoa of cattle bull is 1.04 ± 0.2 mg ⁄ ml and 2.18 ± 0.22 mg ⁄ 109 cells, respectively (Pursel and Graham 1967) Amount of cholesterol in whole semen of cattle bull is 104–412 mg ⁄ 100 ml (RoyChoudhury 1970) Amount of phospholipids in seminal plasma of cattle bull is 1.491 mg ⁄ ml (Jain and Anand 1976) Amount of phospholipids in spermatozoa of cattle bull is 0.416 mg ⁄ 109 cells (Jain and Anand 1976) Amount of phosphatidyl choline in seminal plasma of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 30.0, 26.3 and 24.5 ± 2.2% of total phospholipids, respectively Amount of phosphatidayl choline in spermatozoa of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 35.6, 30.1 and 17.9 ± 0.8% of total phospholipids, respectively Amount of phosphatidayl choline in semi nal plasma of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 23.6%, 17.6% and 32.9 ± 2.0% of total phospholipids, respectively Amount of phosphatidayl choline in spermatozoa obtained from cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 28.0%, 31.8% and 36.8 ± 1.4% of total phospholipids, respectively 556 SMH Andrabi Table Continued Characteristic of component Phosphatidyl ethanolamine Reference Whole semen Seminal plasma Spermatozoa Jain and Anand 1976 11.7 ± 1.5% of total phospholipids 10.8 ± 2.0% of total phospholipids Sarmah et al 1983 10.8 ± 1.4% of total phospholipids 9.3 ± 1.2% of total phospholipids Jain and Anand 1976 4.1 ± 0.3% of total phospholipids 3.4 ± 0.5% of total phospholipids Sarmah et al 1983 4.9 ± 0.7% of total phospholipids 5.7 ± 0.7% of total phospholipids Jain and Anand 1976 13.1 ± 0.7% of total phospholipids 11.3 ± 0.7% of total phospholipids Sarmah et al 1983 13.8 ± 0.8% of total phospholipids 17.4 ± 1.3% of total phospholipids Phosphatidyl serine Jain and Anand 1976 2.8 ± 0.4% of total phospholipids 1.5 ± 0.3% of total phospholipids Phosphatidyl serine + phosphatidyl inositol Sarmah et al 1983 6.1 ± 0.7% of total phospholipids 8.1 ± 0.3% of total phospholipids Phosphatidyl inositol Jain and Anand 1976 2.9 ± 0.5% of total phospholipids 0.6 ± 0.1% of total phospholipids Lysophosphatidyl choline Jain and Anand 1976 3.9 ± 0.9% of total phospholipids 3.9 ± 0.5% of total phospholipids Sarmah et al 1983 3.1 ± 0.2% of total phospholipids 8.3 ± 0.1% of total phospholipids Phosphatidal ethanolamine (ethanolamine plasmogen) Sphinogomyelin Comment Amount of phosphatidyl ethanolamine in seminal plasma of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 10.5, 5.4 and 5.6 ± 0.4% of total phospholipids, respectively Amount of phosphatidyl ethanolamine in spermatozoa of cattle bull which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 20.0%, 9.7% and 5.3 ± 0.4% of total phospholipids, respectively Amount of phosphatidal ethanolamine in seminal plasma of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is16.3%, 5.0% and 9.0 ± 0.9% of total phospholipids, respectively Amount of phosphatidal ethanolamine in spermatozoa of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 7.2%, 4.1% and 9.0 ± 0.4% of total phospholipids, respectively Amount of sphinogomyelin in seminal plasma of cattle bull which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is16.3%, 13.2% and 11.6 ± 1.0 9% of total phospholipids, respectively Amount of sphinogomyelin in cattle bull spermatozoa which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 9.1%, 11.5% and 12.2 ± 1.2% of total phospholipids, respectively Amount of phosphatidyl serine in seminal plasma of cattle bull is 1.3 ± 0.3% of total phospholipids (Jain and Anand 1976) Amount of phosphatidyl serine in spermatozoa of cattle bull is 1.7 ± 0.4% of total phospholipids (Jain and Anand 1976) Amount of Phosphatidyl serine + Phosphatidyl inositol in seminal plasma of cattle bull is 3.6% of total phospholipids (Clegg and Foote 1973) Amount of phosphatidyl serine + phosphatidyl inositol in spermatozoa of cattle bull is 0.7% of total phospholipids (Clegg and Foote 1973) The value of phosphatidyl inositol in seminal plasma obtained in this study differ from that of cattle bull which according to Jain and Anand (1976) is 0.8 ± 0.2% of total phospholipids The value of phosphatidyl inositol in spermatozoa obtained in this study differ from that of cattle bull which according to Jain and Anand (1976) is 1.0 ± 0.2% of total phospholipids Amount of lysophosphatidyl choline in seminal plasma of cattle bull, which according to Clegg and Foote (1973), and Jain and Anand (1976) is 2.2% and 1.2 ± 0.3% of total phospholipids, respectively Amount of lysophosphatidyl choline in spermatozoa of cattle bull which according to Clegg and Foote (1973), and Jain and Anand (1976) is 1.7% and 1.9 ± 0.5% of total phospholipids, respectively Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa 557 Table Continued Characteristic of component Reference Lysophosphatidyl ethanolamine Whole semen Seminal plasma Spermatozoa Comment Jain and Anand 1976 5.6 ± 1.5% of total phospholipids 4.4 ± 1.0% of total phospholipids Sarmah et al 1983 6.6 ± 1.0% of total phospholipids Lysophosphatidyl serine Jain and Anand 1976 1.0 ± 0.3% of total phospholipids 0.7 ± 0.1% of total phospholipids Diphosphatidyl glycerol (cardiolipin) Jain and Anand 1976 7.4 ± 1.3% of total phospholipids 5.5 ± 0.7% of total phospholipids Sarmah et al 1983 3.5 ± 0.5% of total phospholipids 4.9 ± 0.4% of total phospholipids Phosphatidic acid Jain and Anand 1976 0.5 ± 0.2% of total phospholipids 0.3 ± 0.1% of total phospholipids Neutral lipids Jain and Anand 1976 0.439 mg ⁄ ml 0.286 mg ⁄ 109 cells Glycolipids Jain and Anand 1976 0.581 mg ⁄ ml 0.397 mg ⁄ 109 cells Glutathione Jain et al 1990 Aspartic acid Chaudhary and Gangwar 1977 0.395 mM Glutamic acid Chaudhary and Gangwar 1977 4.28 mM Serine Chaudhary and Gangwar 1977 0.60 mM Alanine Chaudhary and Gangwar 1977 0.413 mM Glycine Chaudhary and Gangwar 1977 1.34 mM Lysine Chaudhary and Gangwar 1977 0.133 mM Amount of lysophosphatidyl ethanolamine in seminal plasma of cattle bull, which according to Clegg and Foote (1973), and Jain and Anand (1976) is 2.2% and 1.2 ± 0.3% of total phospholipids, respectively Amount of lysophosphatidyl ethanolamine in spermatozoa of cattle bull which according to Jain and Anand (1976) is 3.2 ± 0.6% of total phospholipids Amount of lysophosphatidyl serine in seminal plasma of cattle bull is 0.4 ± 0.1% of total phospholipids (Jain and Anand 1976) Amount of lysophosphatidyl serine in spermatozoa of cattle bull is 0.5 ± 0.1% of total phospholipids (Jain and Anand 1976) Amount of cardiolipin in seminal plasma of cattle bull, which according to Pursel and Graham (1967), Clegg and Foote (1973), and Jain and Anand (1976) is 5.4%, 8.8% and 5.0 ± 0.5% of total phospholipids, respectively Amount of cardiolipin in spermatozoa of cattle bull, which according to Clegg and Foote (1973), and Jain and Anand (1976) is 6.3% and 5.9 ± 1.0% of total phospholipids, respectively Amount of phosphatidic acid in seminal plasma of cattle bull is 0.4 ± 0.1% of total phospholipids (Jain and Anand 1976) Amount of phosphatidic acid in spermatozoa obtained of cattle bull is 0.2 ± 0.1% of total phospholipids (Jain and Anand 1976) Amount of neutral lipids in seminal plasma of cattle bull is 0.896 mg ⁄ ml (Jain and Anand 1976) Amount of neutral lipids in spermatozoa of cattle bull is 0.164 mg ⁄ 109 cells (Jain and Anand 1976) Amount of glycolipids in seminal plasma of cattle bull is 0.713 mg ⁄ ml (Jain and Anand 1976) Amount of glycolipids in spermatozoa of cattle bull is 0.154 mg ⁄ 109 cells (Jain and Anand 1976) Amount of glutathione obtained in whole semen of cattle bull is 45.35 ± 5.07 lmol ⁄ ml (Jain and Anand 1976) Amount of aspartic acid in seminal plasma of cattle bull is 0.369 ± 0.025 lmoles ⁄ ml (Al-Hakim et al 1970) Amount of glutamic acid in seminal plasma of cattle bull is 4.352 ± 0.257 lmoles ⁄ ml (Al-Hakim et al 1970) Amount of serine in seminal plasma of cattle bull is 0.506 ± 0.03 lmoles ⁄ ml (Al-Hakim et al 1970) Amount of alanine in seminal plasma of cattle is 1.078 ± 0.071 lmoles ⁄ ml (AlHakim et al 1970) Amount of glycine in seminal plasma of cattle bull is 0.564 ± 0.031 lmoles ⁄ ml (Al-Hakim et al 1970) Amount of lysine in seminal plasma of cattle bull is 0.177 ± 0.010 lmoles ⁄ ml (Al-Hakim et al 1970) 32.49 ± 5.10 lmol ⁄ ml Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 558 SMH Andrabi Table Continued Characteristic of component Reference Deoxyribo-nuclease Chauhan et al 1975 Acid phosphatase Chauhan and Srivastava 1973 Dabas et al 1984 Alkaline phosphatase Chauhan and Srivastava 1973 Dabas et al 1984 Whole semen Seminal plasma Spermatozoa 2007.33 ± 112.01 KU ⁄ ml 315.31 ± 22.66 BU ⁄ 100 ml 194 ± 10 BU ⁄ 100 ml 312.50 ± 24.04 BU ⁄ 100 ml 270 ± BU ⁄ 100 ml 39 ± BU ⁄ 1011 cell 63 ± BU ⁄ 1011 cell Comment Amount of deoxyribonuclease in spermatozoa of cattle bull is 1843.4 ± 126.36 KU ⁄ ml (Chauhan et al 1975) Amount of acid phosphatase in seminal plasma and spermatozoa of cattle bull is 182 ± 10 BU ⁄ 100 ml and 25 ± BU ⁄ 1011 cell respectively (Dabas et al 1984) Amount of alkaline phosphatase in seminal plasma and spermatozoa of cattle bull is 246 ± BU ⁄ 100 ml and 54 ± BU ⁄ 1011 cell, respectively (Dabas et al 1984) a Values are mean ± SEM Table Phospholipid composition (% of total phospholipids) of plasma membrane of buffalo and cattle bull spermatozoa Phospholipid Phosphatidyl ethanolamine Phosphatidyl choline Phosphatidyl serine Phosphatidyl inositol Sphinogomyelin Lysophosphatidyl choline Phosphatidyl glycerol Diphosphatidyl glycerol Buffalo (Cheshmedjieva and Dimov 1994) 22.9 ± 66.0 ± 3.5 ± 2.5 ± 8.0 ± – 4.7 ± 5.0 ± 1.6a 3.5 0.5 0.4 1.9 0.6 0.9 Cattle (Parks et al 1987) 9.9 50.3 1.1 2.7 12.6 1.8 – – a Values are mean ± SE Additionally, the differences regarding efficacy of different buffers suggest that buffalo spermatozoa are more prone to freezing stress as compared to cattle bull spermatozoa possibly because of biochemical factors that influence membrane fluidity during cryogenic preservation (refer to Table 4) Therefore, there is a need to study the influence of selected buffers on pre- and postcryogenic membrane stability i.e., in terms of biochemical ⁄ molecular level changes in lipid bilayer and phase transition Permeable cryoprotectant Glycerol is often poly-hydroxylated and capable of hydrogen bonding with water, capable of permeating across the cell membrane, and non-toxic during exposure to cells in the concentration between approximately 1–5 mol ⁄ l, depending on cell type and conditions of exposure (Fuller and Paynter 2004) More specifically, the physiological actions of glycerol during the cryopreservation of spermatozoa take place by replacing intracellular water necessary for the maintenance of cellular volume, interaction with ions and macromolecules, and depressing the freezing point of water and the consequent lowering of electrolyte concentrations in the unfrozen fraction so that less ice forms at any given temperature (Holt 2000b; Medeiros et al 2002) For cryopreservation of buffalo semen, several studies have been carried out in an attempt to find the optimum levels of glycerol and glycerolization In this context, Jainudeen and Das (1982) studied the effect of two glycerolization procedures (one step vs two steps) and the influence of glycerol level in the extender (3%, 5% or 7%) They found that glycerolization procedure had no significant effect on sperm survival traits like motility and acrosomal integrity They also found that post-thaw motility of spermatozoa was significantly better in a 5% glycerol extender, whereas the percentage of intact acrosomes was greater in spermatozoa extended in 3% or 5% glycerol than in spermatozoa extended in 7% glycerol In another study, Kumar et al (1992) found that the best level of glycerol was 6% for Tris- and milk-based diluents, and 9% glycerol for the sodium citrate diluent to obtain better post-thaw motility for buffalo spermatozoa Ramakrishnan and Ariff (1994), and Nastri et al (1994) also tried to reduce the glycerol concentrations from 8% to 2% or 3%, but they found that the reduction in glycerol below 5% decreased the post-thaw motility and ⁄ or acrosome integrity of spermatozoa in the extenders tested Abbas and Andrabi (2002) studied the effects of different concentrations of glycerol (2%, 3%, 4%, 5%, 6%, 7%, 8%, 10% or 12%) on post-thaw sperm quality They reported that the spermatozoa frozen in 7% were significantly better to those in other concentrations of glycerol as judged by post-thaw motility, survivability and plasma membrane integrity Regarding glycerolization, Singh et al (2006) have confirmed that single step is more suitable for the cryopreservation of buffalo spermatozoa in terms of post-thaw forward motility Ethylene glycol could be another option for the cryopreservation of buffalo spermatozoa Permeability of ethylene glycol was found to be higher than glycerol Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa in spermatozoa of different species (Gilmore et al 1995, 1998; Phelps et al 1999), resulting in lower hydraulic conductivity and then in a reduction in the osmotic stress to which cells are exposed during cooling and freezing (Gilmore et al 1995) Propylene glycol also has the basic properties of a cryoprotectant i.e., it is miscible with water in all proportions, its solutions in water have profoundly depressed freezing points, and presumably, it has a low intrinsic toxicity as it is widely used in the food and pharmaceutical industries (Arnaud and Pegg 1990) Recently, Valdez et al (2003) and Rohilla et al (2005) have tested ethylene glycol or propylene glycol as substitute for glycerol Their preliminary results suggest that ethylene glycol may be used for freezing bubaline spermatozoa Therefore, there is a need to study in detail the factors that may affect the viability of frozen buffalo spermatozoa with ethylene glycol as a cryoprotectant Further studies, are also suggested for testing propylene glycol as a cryoprotectant for buffalo spermatozoa Dimethyl sulfoxide (DMSO) is a rapid penetrating cryoprotectants having lower molecular weight than glycerol Also DMSO may inhibit harmful effect of hydroxyl radicals (Yu and Quinn 1994), as these radicals appear during cell respiration and are detrimental to cell (Johnson and Nasr-Esfahani 1994) More recently, Rasul et al (2007) studied glycerol and ⁄ or DMSO, added either at 37°C or at 4°C as a cryoprotectant for buffalo spermatozoa The concentrations (%) of glycerol and DMSO adjusted were : 0, : 1.5, : 3; : 0, : 1.5, : 3; and : 0, : 1.5, : respectively It was, concluded that addition of DMSO at the levels investigated did not improve the post-thaw quality of spermatozoa However, glycerol at a concentration of 6%, when added at 37°C, provided the maximum cryoprotection to the motility apparatus, and plasma membrane integrity of buffalo spermatozoa in Tris– citric acid based extender The exact mechanism involved in the antagonist effect of DMSO on the cryoprotection ability of glycerol is not understood Moreover, the lethal effect of DMSO is attributed to its toxic effect rather than osmotic (Rasul et al 2007) It is believed that because of the lower molecular weight of DMSO, its penetrating ability into the cell is higher than glycerol From the available studies, it is therefore, suggested that a glycerol concentration of 5–7% added initially in the extender may be suitable for the cryopreservation of buffalo bull spermatozoa On the other hand, development of less toxic cryoprotectant could make a significant contribution in improving the quality of frozen–thawed buffalo spermatozoa Non-permeable cryoprotectant Egg yolk is a common component of semen freezing extenders for most of the livestock species, including the buffalo (Sansone et al 2000) It is widely believed that low density lipoproteins (LDL) contained in egg yolk is largely responsible for sperm protection during cryopreservation (Pace and Graham 1974; Watson 1976) It is suggested that LDL adheres to sperm membrane and provides protection to sperm by stabilizing the mem- 559 brane A second hypothesis suggests that phospholipids present in LDL protect sperm by forming a protective film on the sperm surface or by replacing sperm membrane phospholipids that are lost or damaged during the cryopreservation process (Foulkes et al 1980; Quinn et al 1980; Graham and Foote 1987) A third mechanism of protection suggests that LDL seizes the deleterious proteins present in seminal plasma thus improving the freezability of spermatozoa (Bergeron and Manjunath 2006) The exact mechanism by which EY preserves the spermatozoa during freeze–thaw process is unknown (Bathgate et al 2006) Review of literature reveals that little attention has been paid to the level of egg yolk necessary for freezing buffalo semen, and generally it is used at a concentration of 20% in semen extender (Sansone et al 2000; Andrabi et al 2008) Furthermore, the use of egg yolk in higher concentration may have deleterious effects combined with toxicity (amino acid oxidase activity) of dead spermatozoa resulting in lower post-thaw spermatozoal quality (Shannon 1972) The enhanced toxicity associated with increased egg yolk is probably due to the elevated substrate available for hydrogen peroxide formation (Tosic and Walton 1950) In this regard, Sahni and Mohan (1990) studied different levels of egg yolk in extender as a nonpermeable cryoprotectant for buffalo semen The concentration of egg yolk used was 0%, 2%, 5%, 10% or 20% They concluded that the concentration of egg yolk in the extender could be reduced from 20% to 5% without any compromise in post-thaw motility of spermatozoa Kumar et al (1994) studied the effect of different levels of egg yolk (0%, 1%, 5%, 10% and 20%) in Tris-based extender on sperm motility and survival before and after freezing in buffalo They found that the best post-thaw motility and survivability was with 5% yolk Singh et al (1999) studied the effect of different levels of egg yolk on freezability of buffalo semen They found that egg yolk at 10% was superior for freezability with regards to pre-freeze and post-thaw sperm motility It was, also suggested that 10% egg yolk is better in a Tris-based extender for freezing buffalo semen compared to at lower concentration (5%) Recently, Andrabi et al (2008) investigated the use of duck egg yolk, Guinea fowl egg yolk and Indian indigenous hen (Desi) egg yolk in extender for improving the post-thaw quality of buffalo bull spermatozoa, and compared it with commercial hen egg yolk It was concluded that duck egg yolk compared to other avian yolks in extender improves the freezability of buffalo bull spermatozoa as judged by motility, survivability, plasma membrane integrity, intactness of acrosome and head, mid-piece and tail abnormalities In this regard, it is suggested that the improvement or decline in postthaw quality of mammalian spermatozoa with egg yolk of different avian species in freezing extender is attributed to the differences in biochemical composition of the yolks (Trimeche et al 1997; Bathgate et al 2006) Studies investigating the influence of egg yolk from different avian species on Jackass sperm during freeze– thawing have found that the ratio of phosphatidyl ethanolamine : phosphatidyl choline appears to play a role in the level of protection afforded to the sperm Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 560 SMH Andrabi (Trimeche et al 1997) This is of interest to mention that Bathgate et al (2006) reported a significant difference in ratio of phosphatidyl ethanolamine : phosphatidyl choline in chicken egg yolk and duck yolk with a higher ratio in later Therefore, it can be put forward that higher ratio of phosphatidyl ethanolamine : phosphatidyl choline in duck egg yolk may have improved the freezability of buffalo spermatozoa in the study by Andrabi et al (2008) It is, also proposed that supplementation of cryodiluent with quail egg yolk for buffalo bull semen needs to be investigated as the ratio of phosphatidyl ethanolamine : phosphatidyl choline in quail yolk is even higher than duck yolk as reported by Bathgate et al (2006) Finally, as the findings of Andrabi et al (2008) are preliminary, therefore, it is suggested that further studies are required to establish the source and levels of egg yolk in freezing medium for buffalo spermatozoa Polyethylene glycol (PEG) is a non-permeable cryoprotectant that may slow down the process of ice nucleation during cryogenic process, thus protecting the cellular membrane Other protective mechanism by PEG may be due to its coupling with hydrophobic molecules to produce non-ionic surfactants Cheshmedjieva et al (1996) studied the effect of addition of PEG 20 to egg yolk based freezing medium on the cholesterol : phospholipid, sphingomyelin : phosphatidyl choline and unsaturated : saturated fatty acids ratios in buffalo spermatozoa They concluded that PEG 20 added to extender preserved the lipids of frozen buffalo spermatozoa Further studies are required to find out that PEG 20 may be a better option for the cryopreservation of buffalo spermatozoa Sugars that are not capable of diffusing across a plasma membrane, such as lactose, sucrose, raffinose, trehalose or dextrans are also added to the extender as non-permeable cryoprotectant In these instances, the sugars create an osmotic pressure, inducing cell dehydration and therefore, a lower incidence of intracellular ice formation These sugars also interact with the phospholipids in the plasma membrane, reorganizing the membrane which results in sperm that is better suited to surviving the cryopreservation process (Molinia et al 1994; Aisen et al 2002) In early studies, Ahmad and Chaudhry (1980) investigated the lactose or fructose based extenders for cryopreservation of buffalo semen It was found that the diluent comprising 11% lactose and 6% fructose achieved the best results as tested by post-thaw motility and survivability Ala Ud et al (1981) tested the post-thaw motility and survivability of buffalo spermatozoa frozen in homogenized whole milk, Laiciphos (IMV), lactose or citrate-based extender They found that lactose-based extender gave a better protection to sperm during the cryogenic procedure Dhami and Sahni (1993) studied the effect of 1% raffinose in semen diluents (Tris–fructose–yolk–glycerol, egg yolk–citrate–glycerol or lactose–egg yolk–glycerol) on enzyme leakage (lactate dehydrogenase) from buffalo spermatozoa during freezing They found that the postthaw quality of spermatozoa was better with raffinose in Tris-based extender compared to other extenders in terms of release of lactate dehydrogenase Keeping in view the current international trends in disease control, it is possible that extenders having ingredients of animal origin (egg yolk) can be the source of microbes ⁄ bacteria, consequently resulting in the contamination of semen (Bousseau et al 1998; MarcoJimenez et al 2004; de Ruigh et al 2006) In this regard, LDL extracted from egg yolk (indirect use) or lecithin from non-animal source like soya need to be tested as a non-permeable cryoprotectant in extender for deepfreezing of buffalo spermatozoa Antibiotic It is documented that bacteria in semen and their control via addition of antibiotics in freezing diluents may affect the viability or fertility of cryopreserved bovine spermatozoa (Thibier and Guerin 2000; Morrell 2006) Presence of bacteria in the ejaculates can affect fertilization directly (Morrell 2006), by adhering to spermatozoa (Bolton et al 1986; Wolff et al 1993; Diemer et al 1996), impairing their motility (Panangala et al 1981; Kaur et al 1986) and inducing acrosome reaction (El-Mulla et al 1996) Microbes can also have an indirect effect by producing toxins (Morrell 2006) Thus, in the use of AI, it is important to control efficiently the population of microorganisms in the semen Conventionally, benzyl penicillin 1000 IU ⁄ ml and streptomycin sulphate 1000 lg ⁄ ml alone or in combination is commonly added to the freezing diluents of buffalo bull semen (Sansone et al 2000; Akhter et al 2008) Regarding control of bacteriospermia in buffalo bull semen with streptomycin and penicillin (SP), it was found that it is not an effective combination (Gangadhar et al 1986; Aleem et al 1990; Hussain et al 1990; Ali et al 1994; Amin et al 1999) More recently, Ahmed and Greesh (2001) and Ahmed et al (2001a,b) found that bacteria isolated from buffalo bull semen were resistant to penicillin Also SP was deleterious to postthaw quality of spermatozoa They concluded that gentamicin (500 lg ⁄ ml) or amikacin (500 lg ⁄ ml) or norfloxacin (200 lg ⁄ ml) are the antibiotics of choice to be added in extender for efficient preservation of buffalo spermatozoa Recently, Hasan et al (2001) and Akhter et al (2008) investigated the effects of a relatively new antibiotic combination (gentamicin tylosin and linco-spectin, GTLS) in extender on bacterial and spermatozoal quality of preserved spermatozoa They concluded that GTLS is more capable than SP for bacterial control of buffalo bull semen as judged by total aerobic bacterial count and ⁄ or in vitro antibiotic sensitivity Moreover, GTLS is not detrimental to spermatozoal viability of buffalo bull It is relevant to mention that Andrabi et al (2001) have reported a better conception rate with frozen–thawed semen having GTLS compared to SP (55.2% vs 41.66%) It is therefore, suggested that GTLS in extender is more efficient for the preservation of buffalo spermatozoa Further, that testing of wider range of new antibiotic is recommended in cryodiluents for improvement in quality of frozen–thawed buffalo spermatozoa Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa Other additives Keeping in view the poor freezability of bubaline semen attempts have been made to improve the basic buffers developed to minimize the deleterious effects of cryogenic procedures There are few scattered studies that have used additives such as antioxidants, chelating agents, metabolic stimulants, detergents etc for improvement in post-thaw quality of buffalo spermatozoa In this regard, Bhosrekar et al (1990) studied the effect of addition of caffeine or triethanolamine lauryl sulphate to Tris–citric acid-based extender They reported that the addition of the detergent improved the post-thaw spermatozoa motility However, inclusion of caffeine to extender did not made any improvement in motility It is believed that the protective effect of detergents may be exerted directly on the sperm membrane or is mediated through a change in the extending medium such as emulsifying the egg yolk lipids to make them more readily available to the plasmalemma during cryopreservation (Graham et al 1971; Arriola and Foote 1987; Buhr and Pettitt 1996) On other hand, the failure of caffeine to make any improvement is not understood Dhami and Sahni (1993) studied the effect of 0.1% cysteine or 0.1% EDTA (sperm membrane stabilizer and capacitation inhibitor) in semen diluents (Tris– fructose–yolk–glycerol, egg yolk–citrate–glycerol or lactose–egg yolk–glycerol) on enzyme leakage (lactate dehydrogenase) from buffalo spermatozoa during freezing They found that the addition of cysteine or EDTA to the experimental extenders did not improve the postthaw quality of spermatozoa in terms of release of lactate dehydrogenase Singh et al (1996) studied the effect of addition of ascorbic acid in the diluent on the quality of deep frozen buffalo bull semen They found that inclusion of ascorbic acid (2.5 mM) in the semen diluent yielded a significantly higher post-thaw motility and survivability The antioxidant effect of ascorbate is related to direct vitamin E regeneration by reducing the tocopheroxyl radical in the one-electron redox cycle (Packer et al 1979; Dalvit et al 1998) Later on, Kolev (1997) studied the effect of vitamin A, D and E in extender on motility, survivability and acrosomal integrity of cryopreserved buffalo bull spermatozoa It was suggested that vitamin E at 0.3 mg ⁄ ml exhibited the best effects It is wellknown that a-tocopherol inhibits lipid peroxidation (LPO) in biological membranes, acting as a scavenger of lipid peroxyl and alkoxyl radicals, thus preventing oxidative damage in cryopreserved bovine semen (Beconi et al 1991) Fabbrocini et al (2000) suggested that for freezing buffalo spermatozoa, addition of sodium pyruvate (1.25 mM) to the extender resulted in significantly better post-thaw progressive motility and viability The beneficial effect of pyruvate and a-ketoacids is attributed to its antioxidant property Shukla and Misra (2005) studied different antioxidants (a-tocopherol, ascorbic acid or n-propyl gallate) added to Tris-based dilutor for improving freezability of bubaline spermatozoa They found that addition of n-propyl gallate (15 lM) helped in retaining significantly 561 high post-thaw motility and viability of spermatozoa It is noteworthy that propyl gallate is also an antioxidant It protects against oxidation by hydrogen peroxide and oxygen-free radicals, in a catalytic manner by converting hydrogen peroxide into water and oxygen Kumaresan et al (2006) studied the effects of addition of oviductal proteins obtained from various stages of the oestrous cycle to Tris-based extenders on spermatozoa characteristics in buffaloes They found that oviductal proteins differentially affected post-thaw sperm motility, viability, acrosomal integrity, bovine cervical mucus penetration test, hypo-osmotic sperm swelling test and LPO level depending on the region of oviduct and the stage of oestrous cycle at which the proteins were obtained Overall, it was implied that incorporation of oviductal proteins in extender before freezing improved functions and reduced the LPO levels in buffalo spermatozoa during cryopreservation The beneficial actions conveyed by oviductal fluid are presently unknown; however, the identification of catalase in cow oviductal fluid by Lapointe et al (1998) suggests that it may be a mechanism by which the oviductal fluid reduces the damage caused by reactive oxygen species to the spermatozoa Recently, Shukla and Misra (2007) conducted a study to improve buffalo semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ⁄ ml) in routinely used extender They found that incorporation of Bradykinin (2 ng ⁄ ml) in Tris-based extender might be useful in improving the quality of frozen–thawed bubaline spermatozoa as determined by live percentage, motility and plasma membrane integrity The exact mechanism of action of Bradykinin is not yet fully understood From the literature cited in this section, it appears that there are some additives, which have some useful effects in terms of improvement in the quality of frozen– thawed buffalo spermatozoa It is relevant to mention that most of these studies are preliminary Therefore, it is suggested that further research is required to establish their beneficial effects on cryopreservation of buffalo spermatozoa Semen processing It is generally accepted that the cryopreservation process itself reduces more than 50% of the sperm viability (Watson 1979) During this process, the spermatozoa are subjected to chemical ⁄ toxic, osmotic, thermal, and mechanical stresses, which are conspicuous at dilution, cooling, equilibration, or freezing and thawing stage The success of semen cryopreservation depends to a notable degree on dilution rate Originally, semen was diluted to protect spermatozoa during cooling, freezing and thawing, but the rate of dilution was often changed for technical reasons, like to increase the number of females, which could be inseminated with each ejaculate, or to standardize the number of spermatozoa in each dose of frozen–thawed semen (Salamon and Maxwell 2000) In farm animals, the semen has been diluted with specific volumes of extenders or by diluting semen to a specific spermatozoa concentration Dilution rates of Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 562 SMH Andrabi : to : 12 have been successfully used for buffalo semen Perhaps a better of diluting semen, for comparison purposes, is based on the sperm concentration (Purdy 2006) Reports of buffalo spermatozoa with acceptable fertility, was with frozen samples ranging from 120 · 106 to 30 · 106 cells ⁄ ml (Tahir et al 1981; Andrabi et al 2006) After dilution, the semen is cooled to a temperature close to 4°C or 5°C Cooling is a period of adaptation of spermatozoa to reduced metabolism Extended semen is cooled slowly to avoid potential of cold shock Cold shock is believed to impair function of membrane proteins that are necessary for structural integrity or ion metabolism (Watson 2000) Major changes in bovine spermatozoa during this phase occur near 15 to 5°C, and not happen below 0°C (Watson 2000) Rapid cooling reduces the rate of fructose breakdown, oxygen uptake, and ATP synthesis by the sperm, which results in the loss of energy supply and motility (Blackshaw and Salisbury 1957; Wales and White 1959) Furthermore, cold shock may increase calcium uptake by sperm (White, 1993) However, some think that a faster cool will not create problem if the semen is extended in an ideal buffering system (Marshall 1984) It has been empirically determined that cooling cattle bull spermatozoa from body temperature to 5°C performed at a rate of 10°C ⁄ h has minimum deleterious effects (Parks 1997) In this regard, Dhami et al (1992) studied the effect of cooling rates (5, 30, 60 and 120 from 10 to 5°C vs 120 from 28 to 5°C) on the deep freezing of buffalo semen diluted in Tris-based extender Their results suggest that buffalo semen can be frozen successfully after 30 of cooling at 10°C as judged by motility and survivability Regarding equilibration, it is traditionally taken as the total time during which, spermatozoa remain in contact with glycerol before freezing At this stage, glycerol penetrates into the sperm cell to establish a balanced intracellular and extracellular concentration It should not be overlooked that the equilibration includes the concentration balance not only of glycerol, but also of the other osmotically active extender components (Salamon and Maxwell 2000) Therefore, this phenomenon interacts with the type of extender (buffer and cryoprotectant) used and could easily interact with other cryogenic procedures (Marshall 1984) In this regard, Tuli et al (1981b) examined equilibration of buffalo semen diluted in Tris or citric acid-based extender for 2, or h They found that post-thaw sperm survivability was better after h equilibration than after or h Talevi et al (1994) cooled buffalo semen from 28°C to 5°C in 15 and then equilibrated at 5°C for h 45 (fast cooling) or cooled it in h and equilibrated for h (slow cooling) They found that post-thaw sperm motility was significantly higher using the slow than the rapid cooling method Conversely, the rate of cooling had no significant effect on acrosome integrity Dhami et al (1996) conducted a study to determine the relative efficacy of four cooling rates (10 ⁄ 30°C to 5°C; and h each) and two equilibration periods at 5°C (0 and h) for cryopreservation of buffalo ejaculates They concluded that slow cooling of straws from 30 to 5°C for h compared with faster cooling (1 h) or lower initial temperature (10°C) and h of equilibration at 5°C appeared necessary for successful cryopreservation of buffalo semen as determined by survivability and fertility Of considerable importance for the cryopreservation is the cooling ⁄ freezing rate in the critical temperature range ()5 to )50°C) that determines whether the spermatozoa will remain in equilibrium with their extracellular environment or become progressively supercooled with the increasing possibility of intracellular ice formation (Kumar et al 2003) During slow cooling, the dehydration of the spermatozoa can proceed to the point of osmotic equilibrium between intracellular and extracellular space i.e., cellular dehydration will be maximal While raising the cooling rate too much, the dehydration is not fast enough to prevent occurrence of intracellular ice nucleation However, if the cooling rate is within the required values (50–100°C) this results in less excessive intracellular dehydration, less excessive intracellular solute concentrations and less shrinkage of the cells (Mazur 1984; Woelders 1997) Moreover, at optimum cooling ⁄ freezing rates, the spermatozoa remain vulnerable to the unfavourable conditions for a shorter period of time (Woelders 1997) It is worth mentioning that for cattle bull semen currently a freezing rate of ‡40°C is practiced in general for cryopreservation during the critical temperature zone (Anzar et al 2002) Sukhato et al (2001) determined the effects of freezing rate and intermediate plunge temperature (cooling at 10, 20 or 30°C ⁄ each to )40, )80 or )120°C before being plunged into liquid nitrogen) on post-thaw quality and fertility of buffalo spermatozoa They found that cooling ⁄ freezing spermatozoa from to )120°C, either at 20 or 30°C ⁄ yielded better progressive motility and fertility rate Bhosrekar et al (1994) compared the conventional (over liquid nitrogen in static vapour for 10 min) and control (programmable) freezing methods for buffalo bull semen It was concluded that freezing at the rate of 17.32°C ⁄ between +4°C and )40°C with programmable freezer produced better quality frozen semen than the conventional method of freezing More recently, Rasul (2000) examined the effect of freezing rates on post-thaw viability of buffalo spermatozoa extended in Tris–citric acid-based extender The freezing rates examined between and )15°C were or 10°C ⁄ min, whereas the freezing rates investigated between )15 and )80°C were 10, 20 or 30°C ⁄ It was concluded that the different freezing rates tested gave similar results in terms of post-thaw spermatozoal viability as judged by visual and computerized motilities, motion characteristics, plasma membrane integrity and intactness of acrosomal ridge In the freeze–thaw procedure, the warming phase is just as important to the survival of spermatozoa as the freezing phase Spermatozoa that have survived cooling to )196°C still face the challenge of warming and thawing, and thus must traverse twice the critical temperature zone i.e., from )5 to )50°C (Marshall 1984) The thawing effect depends on whether the rate of cooling has been sufficiently high to induce intracellular freezing, or low enough to produce cell dehydration In the former case, fast thawing is required to prevent Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa recrystallization of any intracellular ice present in the spermatozoa Spermatozoa thawed at a fast rate may also be exposed for a shorter time to the concentrated solute and cryoprotectant-glycerol, and the restoration of the intracellular and extracellular equilibrium is more rapid than for slow thawing (Salamon and Maxwell 2000) Also leaving straws in high temperatures for too long time may result in pH fluctuation and subsequently protein denaturation and cell death A practical thaw for cattle bull spermatozoa, recommended by most AI organizations, is as 35°C water bath for at least 30 s (Marshall 1984) For cryopreservation of buffalo spermatozoa in Tris-based extender, Rao et al (1986) tested two thawing rates (37°C for 30 s and 75°C for s) They concluded that the best value for post-thaw motility was observed for semen thawed at 37° for 30 s Dhami et al (1992) studied the effect of thawing rates (40°C for 60 s, 60°C for 15 s and 80°C for s) on post-thaw motility of buffalo spermatozoa cryopreserved in Tris-based extender They reported that thawing at 60°C for 15 s yielded a higher sperm motility compared to other rates In another study, Dhami et al (1996) determined the thawing rates for buffalo semen The thawing rates investigated were 4°C for min, 40°C for or 60°C for 15 s They concluded that thawing of semen at 60°C for 15 s yielded high post-thawing spermatozoal recovery and longevity Sukhato et al (2001) determined the effect of thawing rates on motility and acrosome integrity of buffalo spermatozoa Thawing of spermatozoa was performed at the rate of (rapid) 1000°C or (slow) 200°C ⁄ They concluded that rapid thawing was superior to slow warming The above-mentioned studies demonstrate that an effective cryopreservation procedure for buffalo spermatozoa can be derived by the systematic examination of various cryobiological factors Therefore, from these studies the cryogenic procedures for buffalo semen can be outlined as; cooling from 37 or 39 to 4°C at the rate of 0.2–0.4°C ⁄ min, equilibration, at least h at 4°C, freezing of straws approximately cm above liquid nitrogen for 10–20 min, or by the fast freezing rates (programmable freezing), and thawing at 45–60°C for at least 15 s However, there is still need to improve the processing techniques for cryopreservation of buffalo spermatozoa It is suggested that to devise efficient cooling ⁄ freezing rates for buffalo spermatozoa, studies involving use of a cryomicroscope should be carried out, as this will permit a direct and continuous viewing of spermatozoa, while the temperature is controlled accurately (Medranol et al 2002) Season of semen collection Freezability of buffalo semen can also be affected by the season of collection i.e., by environmental factors like temperature, humidity and day length in a particular season For the first time Tuli and Mehar (1983) studied the seasonal variation in freezability of buffalo semen diluted in Tris-based extender They found that post-thaw spermatozoa motility, significantly increased in winter than summer season After that, Heuer et al (1987) studied the effect of season on in vivo fertility of frozen buffalo semen diluted in chemically defined buffers They 563 reported that semen collected in November (winter) produced significantly higher conception rate than semen collected in June (summer) over a total of 3220 inseminations in both seasons (40.9 vs 34.0%) They attributed 40% of the observed seasonality of buffalo fertility to the male Bhavsar et al (1989a) also studied the monthly variations in freezability and fertility of buffalo bull semen They found that fertility of semen collected, frozen and inseminated during season from July to January (monsoon or late wet summer to autumn and winter) was significantly higher than ejaculates processed and inseminated during the season from February to June (spring to early dry summer) Sagdeo et al (1991) studied the seasonal variations in freezability of buffalo bull semen They found from the data of over a 4-year period that the season significantly affected the post-thaw sperm motility, and values being highest in ejaculates frozen in the winter and lowest in summer Similarly, Bahga and Khokar (1991) studied the seasonal variations in freezability of buffalo bull semen They found that post-thaw semen motility was significantly affected by season of collection, being lowest in summer and highest in winter (December–January) Younis et al (1998) studied the freezability of semen collected during the low breeding season (May–July) and the peak breeding season (September–November) in young (3–4 years), adult (6–8 years) and old (12–15 years) buffalo bulls They reported that post-thaw motility and liveability of spermatozoa frozen in Tris-based extender were significantly higher in adult bulls during the peak breeding season In addition, the sperm abnormalities and deleterious enzymatic activity in frozen–thawed semen were significantly higher during the low breeding season than in the peak breeding season Recently, Koonjaenak et al (2007a) studied the seasonal effect on quality of frozen–thawed buffalo spermatozoa diluted in Tris-based buffer They compared post-thaw sperm quality over three seasons of the year (rainy: July–October; winter: November–February; and summer: March–June), with distinct ambient temperature and humidity Their conclusion was that postthaw plasma membrane integrity and stability were significantly better in ejaculates processed during winter than in samples processed during the other seasons of the year From the above-mentioned studies, it is evident that there is a higher loss of viability and fertility during the process of cryopreservation in summer, thus confirming that vitality of buffalo spermatozoa remain comparatively poorer during this season Moreover, it is suggested that to increase fertility rate in buffalo, semen should be collected and preserved during cooler months and used for AI all over the year In another study, Koonjaenak et al (2007b) investigated the frozen–thawed buffalo sperm nuclear DNA fragmentation by flowcytometry and head morphology over three seasons in tropical Thailand (the rainy season, July–October; winter, November–February; and summer, March–June) They found that the DNA fragmentation index (DFI) values varied statistically among seasons, being lower in the rainy season than in winter or summer, and were affected by the year of semen collection and processing The proportion of morphologically Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 564 SMH Andrabi abnormal sperm head shapes was low, with no significant differences between seasons However, DFI was significantly related to the proportion of loose abnormal sperm heads It was concluded that frozen–thawed buffalo sperm chromatin is not critically damaged by cryopreservation or affected by the seasonal variations in temperature and humidity seen in tropical Thailand There is 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 2007a) Recently, Argov et al (2007) have reported that cattle semen samples collected during the summer and considered to be of good quality had alterations in lipid concentration, fatty-acid composition and cholesterol level In addition, they provided the first evidence for the existence of a very-low-density lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular lipids Interestingly, the expression of VLDLr was threefold greater in samples collected during the winter than in those collected in the summer Therefore, it is suggested that such modifications may explain, in part, the reduced freezability of buffalo semen collected during the summer Few scattered reports are available that describe the 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) However, the information given in these studies are insufficient to explain the variation in freezability of buffalo spermatozoa during the different seasons Therefore, detailed studies should be carried out to ascertain the biochemical or structural differences in seminal plasma, spermatozoa, or plasmalemma, which might be influencing the freezability of buffalo spermatozoa during the different months ⁄ seasons Conclusions Viability and fertility of frozen–thawed buffalo bull spermatozoa is considerably lower than that of cattle bull Several buffers, cryoprotectants, antibiotics, other agents and various cooling, freezing and thawing rates initially developed for cattle bull spermatozoa have been used, at times with contrasting results Therefore, a better understanding of the fundamental principle of cryopreservation of buffalo spermatozoa is necessary according to the specific requirements Moreover, there is a need to develop biochemically defined extenders and cryogenic procedures that may result in improvement in viability and fertility of frozen–thawed buffalo spermatozoa Besides this, the season during which the semen is collected should also be considered as a variable affecting quality of cryopreserved buffalo spermatozoa References Abbas A, Andrabi SMH, 2002: Effect of different glycerol concentrations on motility before and after freezing, recovery rate, longevity and plasma membrane integrity of NiliRavi buffalo bull spermatozoa Pak Vet J 22, 1–4 Ahmad K, Chaudhry RA, 1980: Cryopreservation of buffalo semen Vet Rec 106, 199–201 Ahmad M, Ahmad KM, Khan A, 1986: Cryopreservation of buffalo spermatozoa in Tris (hydroxymethyl-aminomethane) Pak Vet J 6, 1–3 Ahmad KM, Khan A, Ahmad M, 1988: Fertility of Nili-Ravi buffalo bull frozen semen at different thawing temperatures Buffalo J 4, 51–55 Ahmad Z, Anzar M, Shahab M, Ahmad N, Andrabi SMH, 2003: Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates Theriogenology 59, 1189–1202 Ahmed K, Greesh M, 2001: Effect of antibiotics on the bacterial load and quality of semen of Murrah buffalo bulls during preservation Indian J Anim Reprod 22, 79–80 Ahmed K, Greesh M, Tripathi RP, 2001a: Effect of different antibiotics on semen quality during post thaw incubation at 37°C Indian J Anim Reprod 22, 81 Ahmed K, Kumar AA, Mohan G, 2001b: Bacterial flora of preputial washing and semen of Murrah buffalo bulls and their antibiotic sensitivity pattern Ind J Comp Microbiol Immunol Infect Dis 22, 63–64 Aisen EG, Medina VH, Venturino A, 2002: Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations Theriogenology 57, 1801–1808 Akhter S, Ansari MS, Andrabi SMH, Ullah N, Qayyum M, 2007: Effect of antibiotics in extender on fertility of liquid buffalo bull semen Pak Vet J 27, 13–16 Akhter S, Ansari MS, Andrabi SMH, Ullah N, Qayyum M, 2008: Effect of antibiotics in extender on bacterial and spermatozoal quality of cooled buffalo (Bubalus bubalis) bull semen Reprod Domest Anim 43, 272–278 Ala Ud D, Chaudhry RA, Ahmad K, 1981: Effect of different extenders on freezability of buffalo semen Pak Vet J 1, 59–61 Aleem M, Chaudhry RA, Khan NU, Rizvi AR, Ahmed R, 1990: Occurrence of pathogenic bacteria in buffalo semen Buffalo J 6, 93–98 Al-Hakim MK, Graham EF, Schmehl ML, 1970: Free amino acids and amino compounds in bovine seminal plasma J Dairy Sci 53, 84–88 Ali H, Din A, Samad HA, Ali S, Sabri MA, 1994: Comparative effects of combiotic, ampicillin and gentamycin sulphate on motility percentage, liveability and absolute index of liveability in the buffalo bull semen Pak Vet J 14, 223–227 Amin AS, Darwish GM, Maha SZ, Hassan HM, 1999: Trial to control Chlamydia psittaci in processed buffalo-semen Assi Vet Med J 40, 319–332 Andrabi SMH, Ahmad N, Abbas A, Anzar M, 2001: Effect of two different antibiotic combinations on fertility of frozen buffalo and Sahiwal bull semen Pak Vet J 21, 166–169 Andrabi SMH, Siddique M, Ullah N, Khan LA, 2006: Effect of reducing sperm numbers per insemination dose on fertility of cryopreserved buffalo bull semen Pak Vet J 26, 17–19 Andrabi SMH, Ansari MS, Ullah N, Anwar M, Mehmood A, Akhter S, 2008: Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa Anim Reprod Sci 104, 427–433 Anzar M, He L, Buhr MM, Kroetsch TG, Pauls KP, 2002: Sperm apoptosis in fresh and cryopreserved bull semen detected by flow cytometry and its relationship with fertility Biol Reprod 66, 354–360 Anzar M, Farooq U, Mirza MA, Shahab M, Ahmad N, 2003: Factors affecting the efficiency of artificial insemination in cattle and buffalo in Punjab, Pakistan Pak Vet J 23, 106– 113 Argov N, Sklan D, Zeron Y, Roth Z, 2007: Association between seasonal changes in fatty-acid composition, Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa expression of VLDL receptor and bovine sperm quality Theriogenology 67, 878–885 Arnaud FG, Pegg DE, 1990: Permeation of glycerol and propane-1,2-diol into human platelets Cryobiology 27, 107– 118 Arriola J, Foote RH, 1987: Glycerolation and thawing effects on bull spermatozoa frozen in detergent-treated egg yolk and whole egg extenders J Dairy Sci 70, 1664– 1670 Bahga CS, Khokar BS, 1991: Effect of different seasons on concentration of plasma luteinizing hormone and seminal quality vis-a-vis freezability of buffalo bulls (Bubalus bubalis) Int J Biometeorol 35, 222–224 Bandyopadhyay SK, Roy DJ, 1975: Freezing and fertilising ability of spermatozoa Indian J Anim Health 14, 37–41 Banerjee AK, Ganguli NC, 1973: Some aspects of the composition of buffalo semen J Reprod Fertil 33, 171–173 Barile VL, Galasso A, Pacelli C, Francillo M, Cigliano A, Penna L, Panfili M, Fiorini M, Borghese A, 1999: Conception rate in synchronized and artificially inseminated buffalo cows in two different seasons under field conditions In: Piva G, Bertoni G, Masoero F, Bani P, Calamari L (ed), Proceedings of XIII National ASPA Cong., Piacenza, Italy Scientific Association of Animal Production, Bologna, Italy, pp 262–264 Barnabe VH, Baruselli P, Barnabe RC, Silva EOTR, Visintin JA, 1994: Artificial insemination of buffaloes using two different diluents In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong., Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 546–548 Basirov EB, 1964: The biology of reproduction and artificial insemination of buffaloes Proceedings of 5th Int Cong Anim Reprod A.I., Trento, Italy International Congress on Animal Reproduction and Artificial Insemination, Milan, Italy, pp 4–10 Bates RG, 1961: Amine buffers for pH control Ann N Y Acad Sci 92, 341–356 Bathgate R, Maxwell WMC, Evans G, 2006: Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post-thaw sperm quality Reprod Domest Anim 41, 68–73 Beconi M, Affranchino M, Schang LM, Beorlegui NM, 1991: Influence of antioxidants on SOD activity in bovine sperm Biochem Int 3, 545–553 Bergeron A, Manjunath P, 2006: New insights towards understanding the mechanisms of sperm protection by egg yolk and milk Mol Reprod Dev 73, 1338–1344 Bhavsar BK, Patel KS, Dhami AJ, Kodagali SB, 1988: Pelleting buffalo semen and its fertility trials Indian J Anim Prod 9, 18–20 Bhavsar BK, Dhami AJ, Kodagali SB, 1989a: Monthly variations in freezability and fertility of Mehsana buffalo semen Indian J Dairy Sci 42, 246–250 Bhavsar BK, Dhami AJ, Kodagali SB, 1989b: Role of semen additives in improving fertility rates of Mehsana buffalo semen Indian Vet J 66, 623–625 Bhosrekar MR, Nagarcenkar R, 1971: Investigations on Semen Quality and Reproductive Behaviour of Buffalo Bulls Annual Report National Dairy Research Institute, Karnal, India, pp 50–51 Bhosrekar MR, Purohit JR, Mangurkar BR, 1990: Studies on the effect of additives to semen diluent Indian J Anim Reprod 11, 85–88 Bhosrekar MR, Mokashi SP, Purohit JR, Gokhale SB, Mangurkar BR, 1994: Effect of glycerolization and deep freezing on the levels and release of enzymes in buffalo semen in relation to initial seminal attributes In: Vale WG, 565 Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong., Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 465–467 Blackshaw AW, Salisbury GW, 1957: Factors influencing metabolic activity of bull spermatozoa 11: cold-shock and its prevention J Dairy Sci 40, 1099–1106 Blesbois E, 2007: Current status in avian semen cryopreservation World’s Poult Sci J 63, 213–222 Bolton VN, Warren RE, Braude PR, 1986: Removal of bacterial contaminants from semen for in vitro fertilization or artificial insemination by the use of buoyant density centrifugation Fertil Steril 6, 1128–1132 Bousseau S, Brillard JP, Marquant L, Guienne B, Guerin B, Camus A, Lechat M, 1998: Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents Theriogenology, 50, 699–706 Buhr MM, Pettitt MJ, 1996: Frozen-thawed boar sperm: isolation of membranes and fluidity measurement Reprod Domest Anim 31, 147–152 Cabrera F, Gonzalez F, Batista M, Calero P, Medrano A, Gracia A, 2005: The effect of removal of seminal plasma, egg yolk level and season on sperm freezability of canary buck (Capra hircus) Reprod Domest Anim 40, 191–195 Chachur MGM, Oba E, Gonzales CIM, 1997: Equilibrium time influence on motility, vigour and membrane integrity of thawed buffalo semen using triladyl, glycine-egg yolk and tes extenders In: Borghese A, Failla S, Barile VL (eds) Proceedings of 5th World Buffalo Cong., Caserta, Italy International Buffalo Federation, Roma, Italy, pp 846–849 Chaudhary KC, Gangwar PC, 1977: Nitrogenous constituents and free amino acid content in the seminal plasma of buffalo (Bos bubalis) in different periods of the year in relation to fertility Zbl Vet Med A 24, 629–635 Chauhan RAS, Srivastava RK, 1973: Enzyme composition of buffalo seminal plasma J Reprod Fert 34, 165–166 Chauhan FS, Dwarkanath PK, Vyas KK, 1975: Deoxyribonuclease in buffalo and cattle bull semen Indian Vet J, 52, 12–13 Cheshmedjieva SB, Dimov VN, 1994: Effect of freezing on phospholipid distribution of buffalo spermatozoa plasma membranes In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong., Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 519–521 Cheshmedjieva SB, Vaisberg CN, Kolev SI, 1996: On the lipid composition of buffalo spermatozoa frozen in different cryoprotective media Bulgarian J Agric Sci 2, 23–26 Chinnaiya GP, Ganguli NC, 1980a: Acrosomal damage of buffalo spermatozoa during freezing in extenders Zbl Vet Med A 27, 339–342 Chinnaiya GP, Ganguli NC, 1980b: Freezability of buffalo semen in different extenders Zbl Vet Med A 27, 563–568 Chinnaiya GP, Sarma PA, Ganguli NC, 1979: Fertility studies on frozen buffalo semen Indian J Dairy Sci 32, 93–94 Chohan KR, Iqbal J, Asghar AA, Chaudhary MA, 1992: Fertility of liquid and frozen semen in Nili-Ravi buffaloes Pak Vet J 12, 4–5 Clegg ED, Foote RH, 1973: Phospholipid composition of bovine sperm fractions, seminal plasma and cytoplasmic droplets J Reprod Fert 34, 379–383 Cockrill WR, 1974: The Husbandry and Health of the Domestic Buffalo Food and Agricultural Organization (FAO), Rome, Italy Dabas YPS, Verma MC, Tripathi SS, Saxena VB, 1984: Comparative study of biochemical constituents of bull and Murrah bull semen Indian J Anim Res 18, 121–123 Dalvit GC, Cetica PD, Beconi MT, 1998: Effect of a-tocopherol and ascorbic acid on bovinein vitro fertilization Theriogenology 49, 619–627 Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 566 SMH Andrabi Dhami AJ, Kodagali SB, 1990: Freezability, enzyme leakage and fertility of buffalo spermatozoa in relation to the quality of semen ejaculates and extenders Theriogenology 34, 853– 863 Dhami AJ, Kodagali SB, 1991: Freezability, fertility and spermatozoan losses in Surti buffalo bulls Indian J Anim Res 24, 39–43 Dhami AJ, Sahni KL, 1993: Effect of extenders, additives and deep freezing on the leakage of lactic dehydrogenase from cattle and buffalo spermatozoa Indian J Anim Sci 63, 251– 256 Dhami AJ, Sahni KL, Mohan G, 1992: Effect of various cooling rates (from 30 deg to 5°C) and thawing temperatures on the deep-freezing of Bos taurus and Bos bubalis semen Theriogenology 38, 565–574 Dhami AJ, Jani VR, Mohan G, Sahni KL, 1994: Effect of extenders and additives on freezability, post-thaw thermoresistence and fertility of frozen Murrah buffalo semen under tropical climate Buffalo J 10, 35–45 Dhami AJ, Sahni KL, Mohan G, Jani VR, 1996: Effects of different variables on the freezability, post-thaw longevity and fertility of buffalo spermatozoa in the tropics Theriogenology 46, 109–120 Diemer T, Weidner W, Michelmann HW, Schiefer HG, Rovan E, Mayer F, 1996: Influence of Escherichia coli on motility parameters of human spermatozoa in vitro Int J Androl 19, 271–277 Drost M, 2007: Bubaline versus bovine reproduction Theriogenology 68, 447–449 El-Mulla KF, Kohn FM, Dandal M, 1996: In vitro effect of Escherichia coli on human sperm acrosome reaction Arch Androl 37, 73–78 European Regional Focal Point on Animal Genetic Resources, 2003: Guidelines for the constitution of national cryopreservation programmes for farm animals In: Hiemstra SJ (ed.), Publication No of the European Regional Focal Point on Animal Genetic Resources Lelystad, Netherlands, p 30 Fabbrocini A, Del Sorbo C, Fasano G, Sansone G, 2000: Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B bubalis) spermatozoa Theriogenology 54, 193– 207 Food and Agricultural Organization (FAO), 2004: FAOSTAT, Global Livestock Production and Health Atlas Animal Production and Health Division, Rome, Italy Available at: (http://www.fao.org/ag/aga/glipha/index.jsp) Foulkes JA, Sweasey D, Goodey RG, 1980: Fertility of bull spermatozoa in egg-yolk diluents of varied lipid fatty acid composition J Reprod Fert 60, 165–169 Fuller B, Paynter S, 2004: Fundamentals of cryobiology in reproductive medicine Reprod Biomed Online 9, 680–691 Gangadhar KS, Rao AR, Subbaiah G, 1986: Effect of antibiotics on bacterial load in frozen semen of buffalo bulls Indian Vet J 63, 489–493 Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK, 1995: Effect of cryoprotectant solutes on water permeability of human spermatozoa Biol Reprod 53, 985–995 Gilmore JA, Liu J, Gao DY, Peter AT, Critser JK, 1998: Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation Biol Reprod 58, 28–36 Gokhale SB, Bhagat RL, 2000: Status of reproductive performance in rural buffaloes artificially inseminated using deep frozen semen Indian J Anim Sci 70, 366–368 Good NE, Izawa S, 1972: Hydrogen ion buffers Methods Enzymol 24, 53–68 Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RMM, 1966: Hydrogen ion buffers for biological research Biochemistry 5, 467–477 Graham JK, Foote RH, 1987: Effect of several lipids, fatty acyl chain length and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing Cryobiology 24, 42–52 Graham EF, Rajamannan AHJ, Schmehl MKL, Maki-Lanrila M, Bower RE, 1971: Preliminary report on procedure and rationale for freezing boar semen AI Digest 19, 12 Graham EF, Crabo BG, Brown KI, 1972: Effect of some zwitterion buffers on the freezing and storage of spermatozoa I Bull J Dairy Sci 55, 372–378 Haranath GB, Suryaprakasam TB, Rao AVN, Somasekharam G, 1990: Freezability of semen and fertility of frozen semen packaged in mini and medium French straws: a note In: Acharya RM, Lokeshwar RR, Kumar AT (eds) Proceedings of 2nd World Buffalo Cong, New Delhi, India International Buffalo Federation, Roma, Italy, pp 87–88 Hasan S, Andrabi SMH, Muneer R, Anzar M, Ahmad N, 2001: Effects of a new antibiotic combination on post-thaw motion characteristics and membrane integrity of buffalo and Sahiwal bull spermatozoa and on the bacteriological quality of their semen Pak Vet J 21, 6–12 Hassan MM, Zia Ur R, 1994: Some factors limiting artificial insemination programme Pak Vet J 14, 115–118 Heuer C, Bajwa MA, 1986: Selection of Nili-Ravi buffaloes for artificial insemination Zuchthygiene 21, 257–262 Heuer C, Tahir MN, Amjad H, 1987: Effect of season on fertility of frozen buffalo semen Anim Reprod Sci 13, 15– 21 Holt WV, 2000a: Fundamental aspects of sperm cryobiology: the importance of species and individual differences Theriogenology 53, 47–58 Holt WV, 2000b: Basic aspects of frozen storage of semen Anim Reprod Sci 62, 3–22 Hussain SS, Ahmad N, Din A, Chaudhry NA, 1990: Effect of different antibiotics on motility and liveability of spermatozoa and viable bacterial count in buffalo semen Pak Vet J 10, 171–174 Jain MC, 1987: Ascorbic acid concentration in bovine and bubaline semen Arch Exp Vet Med Leipzig 41, 550– 555 Jain YC, Anand SR, 1976: The lipids of buffalo spermatozoa and seminal plasma J Reprod Fert 47, 255–260 Jain MC, Arora N, Jogi S, Mishra DD, 1990: Biochemical studies into variation and repeatability of glutathione concentrations in bovine and bubaline semen Arch Exp Vet Med Leipzig 44, 569–571 Jainudeen MR, Das S, 1982: Effect of level of glycerol, rates of freezing and thawing on the survival of buffalo spermatozoa in straws In: Jainudeen MR, Omar AR (eds), Proceedings of Asian-Australian Anim Sci Cong, Serdang, Malaysia Penerbit University, Kuala Lumpur, Malaysia, pp 409–411 Johnson MH, Nasr-Esfahani MH, 1994: Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? Bioessays 16, 31–38 Kakar SS, Anand SR, 1981: Changes in adenosine 5¢-triphosphate, adenylate energy charge and adenosine 3¢5¢-cyclic monophosphate during the freezing of buffalo semen J Reprod Fert 62, 543–548 Kanchan, Singh N, 2005: Semen characteristics, semen discard rate and fertility in Murrah buffalo bulls Indian J Anim Reprod 26, 120–122 Kaur M, Tripathi KK, Bansal MR, Jain PK, Gupta KG, 1986: Bacteriology of cervix incases of infertility: effect on human sperm American J Reprod Immunol Microbiol 12, 21–24 Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa Keith JE, Morrison JF, 1981: Buffers of constant ionic strength for studying pH-dependent processes Methods Enzymol 87, 405–426 Kolev SI, 1997: Effect of vitamins A, D, E on the motility and acrosomal integrity of cryopreserved buffalo bulls’ spermatozoa In: Borghese A, Failla S, Barile VL (eds), Proceedings of 5th World Buffalo Cong., Caserta, Italy International Buffalo Federation, Roma, Italy, pp 833– 835 Koonjaenak S, Pongpeng P, Wirojwuthikul S, Johannisson A, Kunavongkrit A, Rodriguez-Martinez H, 2007a: Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis) Theriogenology 67, 1424–1435 Koonjaenak S, Johannisson A, Pongpeng P, Wirojwuthikul S, Kunavongkrit A, Rodriguez-Martinez H, 2007b: Seasonal variation in nuclear DNA integrity of frozen–thawed spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis) J Vet Med A 54, 377–383 Kumar S, Sahni KL, Mohan G, 1992: Effect of different levels of glycerol and yolk on freezing and storage of buffalo semen in milk, Tris and sodium citrate buffers Buffalo J 8, 151–156 Kumar S, Sahni KL, Mohan G, 1994: Effect of yolk, glycerol and sugars on post-thaw survival of buffalo spermatozoa in Tris dilutor Indian J Anim Sci 64, 362–364 Kumar S, Millar JD, Watson PF, 2003: The effect of cooling rate on the survival of cryopreserved bull, ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines Cryobiology 46, 246–253 Kumaresan A, Ansari MR, Abhishek G, 2005: Modulation of post-thaw sperm functions with oviductal proteins in buffaloes Anim Reprod Sci 90, 73–84 Kumaresan A, Ansari MR, Garg A, Kataria M, 2006: Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes Anim Reprod Sci 93, 246–257 Lapointe S, Sullivan R, Sirard MA, 1998: Binding of a bovine oviductal fluid catalase to mammalian spermatozoa Biol Reprod 58, 747–753 Marco-Jimenez F, Puchades S, Moce E, Viudes-de-Cartro MP, Vicente JS, Rodriguez M, 2004: Use of powdered egg yolk vs fresh egg yolk for the cryopreservation of ovine semen Reprod Domest Anim 39, 438–441 Marshall CE, 1984: Considerations for cryopreservation of semen Zoo Biol 3, 343–356 Matharoo JS, Garcha DS, 1986: A note on the reproductive performance of buffalo bulls under field conditions Livestock Adv 11, 37–39 Matharoo JS, Singh M, 1980: Revivability of buffalo-spermatozoa after deep freezing the semen using various extenders Zbl Vet Med A 27, 385–391 Mazur P, 1984: Freezing of living cells: mechanisms and implications Am J Physiol 247, C125–C142 Medeiros CMO, Forell F, Oliveira ATD, Rodrigues JL, 2002: Current status of sperm cryopreservation: why is it better Theriogenology 57, 327–344 Medranol A, Watson PF, Holt WV, 2002: Importance of cooling rate and animal variability for boar sperm cryopreservation: insights from the cryomicroscope Reproduction 123, 315–322 Mohan G, Madan ML, Razdan MN, 1979: Composition of Murrah buffalo bull semen during winter and summer months in India Trop Agric (Trinidad) 54, 21–28 Molinia FC, Evans G, Casares PI, Maxwell WMC, 1994: Effect of monosaccharides and disaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa Anim Reprod Sci 36, 113– 122 567 Morrell JM, 2006: Update on semen technologies for animal breeding Reprod Domest Anim 41, 63–67 Morris GJ, Clarke AG, 1987: Cells at low temperatures In: Grout BWW, Morris GJ (eds), The Effects of Low Temperatures on Biological Systems Edward Arnold, London, pp 72–119 Muer SK, Roy SB, Mohan G, Dhoble RL, 1988: Cryogenic changes in seminal protein of cattle and buffalo Theriogenology 30, 1005–1010 Nastri MJF, Del Sorbo C, Fabbrocini A, Fasano G, Sansone G, 1994: Performances motility in cooled and freeze thawed B bubalis spermatozoa at different osmotic pressures In: Bradley M, Cummins JM (eds), Proceeding of 7th Int Symp Spermatology, Cairns, Australia Australian Society of Reproductive Biology, Victoria, Australia, pp 99–100 Oba E, Fuck EJ, Bicudo SD, Pap FO, Ohashi OM, 1994: Prelimanary study on different mediums for deep freezing of buffalo semen In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong., Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 579–581 Pace MM, Graham EF, 1974: Components in egg yolk which protect bovine spermatozoa during freezing J Anim Sci 39, 1144–1149 Packer JE, Slater TF, Wilson RL, 1979: Direct observation of a free radical interaction between vitamin E and vitamin C Nature 278, 737–738 Panangala VS, Winter AJ, Wijesinha A, Foote RH, 1981: Decreased motility of bull spermatozoa caused by Mycoplasma bovigenitalium Am J Vet Res 42, 2090–2093 Pant HC, Barot LR, Kasiraj R, Misra AK, Prabhakar JH, 2001: Effect of clitoral stimulation after artificial insemination on conception rate in the buffalo Bubalus Bubalis 7, 66–69 Parks JE, 1997: Hypothermia and mammalian gametes In: Karow AM, Critser JK (eds), Reproductive Tissue Banking Academic Press, San Diego, CA, pp 229–261 Parks JE, Arion JA, Foote RH, 1987: Lipids of plasma memebrane and outer acrosomal membrane from bovine spermatozoa Biol Reprod 37, 1249–1258 Phelps MJ, Liu J, Benson JD, Willoughby CE, Gilmore JA, Critser JK, 1999: Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation Biol Reprod 61, 1031–1041 Prabhakar JH, Kalsi JS, Jani VR, Patel SH, 2002: Effect of live sperm count per inseminate on pregnancy rate in buffaloes Indian J Anim Reprod 23, 170–172 Presicce GA, Verberckmoes S, Senatore EM, Pascale M, Dewulf JJ, Soom AV, 2004: Assessment of a new uterotubal junction insemination device in the Mediterranean Italian water buffalo (Bubalus bubalis) under field conditions Bubalus Bubalis 10, 58–64 Purdy PH, 2006: A review on goat sperm cryopreservation Small Rumin Res 63, 215–225 Pursel VG, Graham EF, 1967: Phospholipids of bovine spermatozoa and seminal plasma J Reprod Fert 14, 203–211 Quinn PJ, Chow PY, White IG, 1980: Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site J Reprod Fertil 60, 403–407 Raizada BC, Sattar A, Pandey MD, 1990: A comparative study of freezing buffalo semen in two dilutors In: Acharya RM, Lokeshwar RR, Kumar AT (eds), Proceedings of 2nd World Buffalo Cong, New Delhi, India International Buffalo Federation, Roma, Italy, pp 66–74 Ramakrishnan P, Ariff MO, 1994: Effect of glycerol level and cooling rate on post-thaw semen quality of Malaysian swamp buffalo In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong, Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 540–542 Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag 568 SMH Andrabi Rao AVN, Haranath GB, Sekharam GS, Rao JR, 1986: Effect of thaw rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws Anim Reprod Sci 12, 123–129 Rasul Z, 2000: Cryopreservation of buffalo semen PhD Thesis, Quaid-i-Azam University, Islamabad, Pakistan Rasul Z, Anzar M, Jalali S, Ahmad N, 2000: Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa Anim Reprod Sci 59, 31–41 Rasul Z, Ahmed N, Anzar M, 2007: Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm Theriogenology 68, 813–819 Rattan PJS, Rao MB, Krishnan CPA, 1980: Biochemical studies on buffalo-bull (Bos bubalis) semen Indian Vet Med J 4, 65–70 Rohilla RK, Tuli RK, Goyal RL, 2005: Comparative study of the effects of cryoprotective agents in freezing Murrah buffalo bull semen Indian J Vet Res 14, 37–43 Roy A, Srivastava RK, Pandey MD, 1956: Deep freezing of buffalo semen diluted and preserved in glycine-egg yolk medium Indian J Dairy Sci 9, 61–62 RoyChoudhury PN, 1970: Total cholesterol content in bull semen Indian Vet J 47, 146–150 de Ruigh L, Bosch JC, Brus MC, Landman B, Merton JS, 2006: Ways to improve the biosecurity of bovine semen Reprod Domest Anim 41, 268–274 Saacke RG, 1984: Semen quality: importance of and influencing factors In: Proceedings of 10th NAAB Tech Conf A I Reprod Milwaukee, WI, USA National Association of Animal Breeders, Columbia, USA, pp 30–36 Sagdeo LR, Chitnis AB, Kaikini AS, 1991: Effect of seasonal variations on freezability of Surti buffalo bull semen Indian J Anim Reprod 12, 1–3 Sahni KL, Mohan G, 1990: Yolk as a cryoprotectant in deep-freezing of bovine semen Indian J Anim Sci 60, 828– 829 Salamon S, Maxwell WMC, 2000: Storage of ram semen Anim Reprod Sci 62, 77–111 Salem HM, Osman SA, 1972: Seasonal variations in the chemical constituents of blood and semen in buffalo and cattle Alexandria J Agric Res 20, 51–55 Sansone G, Nastri MJF, Fabbrocini A, 2000: Storage of buffalo (Bubalus bubalis) semen Anim Reprod Sci 62, 55– 76 Sarmah BC, Kaker ML, Razdan MN, 1983: Total lipids and phospholipids in buffalo semen (Bubalus bubalis) Theriogenology 20, 521–527 Senatore EM, Verberckmoes S, Pascale M, Presicce GA, 2004: A deep utero-tubal semen deposition in Mediterranean Italian buffaloes using a new artificial insemination device Reprod Fertil Dev 16, 133 Shannon P, 1972: The effect of egg yolk level and dose rate of semen diluted in caprogen Proceedings of 7th Int Cong Anim Reprod A.I Munich, Germany International Congress on Animal Reproduction and Artificial Insemination, Milan, Italy, pp 1440–1442 Shukla MK, Misra AK, 2005: Effect of antioxidants alpha tocopherol, ascorbic acid and n-propyl gallate on Murrah semen cryopreservation Buffalo J 21, 27–38 Shukla MK, Misra AK, 2007: Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation Anim Reprod Sci 97, 175–179 Sidhu KS, Guraya SS, 1979: Effects of seasons on physiochemical characteristics of buffalo (Bubalus bubalis) semen Indian J Anim Sci 49, 884–889 Singh B, 1990: Sampling methodology for the study of effectiveness of artificial insemination in buffaloes under field conditions Indian J Anim Sci 60, 773–776 Singh M, Pant HC, 2000: Effect of post-thaw incubation on semen quality of buffalo bulls – comparison with cattle Buffalo Bull 19, 51–54 Singh B, Singh D, 1988: Factors affecting conception rate in buffalo through artificial insemination under field conditions Indian J Anim Sci 58, 798–799 Singh B, Mahapatro BB, Sadhu DP, 1969: Chemical composition of cattle and buffalo spermatozoa and seminal plasma under different climatic conditions J Reprod Fert 20, 175– 178 Singh M, Matharoo JS, Chauhan FS, 1980: Preliminary fertility results with frozen buffalo bull semen using Tris extender Theriogenology 13, 191–194 Singh J, Pangawkar GR, Biswas RK, Srivastava AK, Sharma RD, 1990: Studies on lactic dehydrogenase and sorbitol dehydrogenase release in relation to deep freezing of buffalo semen in certain extenders Theriogenology 34, 371–378 Singh J, Pangawkar GR, Biswas RK, Kumar N, 1991: Studies on buffalo sperm morphology during various stages of freezing in certain extenders Indian J Anim Reprod 12, 126–129 Singh B, Chand D, Singh P, Yadav NK, 1996: Effect of vitamin C addition in the diluent on the quality of deep frozen Murrah buffalo bull (Bubalus bubalis) semen Int J Anim Sci 11, 131–132 Singh P, Singh S, Hooda OK, 1999: Effect of different level of egg yolk on freezability of buffalo semen Haryana Vet 38, 26–28 Singh P, Jindal JK, Singh S, Hooda OK, 2000: Freezability of buffalo bull semen using different extenders Indian J Anim Reprod 21, 41–42 Singh P, Singh I, Singh S, Sharma RK, 2006: Initial stage glycerolization prevents the incidence of backward sperm motility during cryopreservation and increases buffalo semen freezability Indian J Anim Sci 76, 777–779 Sosa GA, El-Deeb ED, El-Sabagh KM, 2003: Interactions of diluents, cryoprotective agents and straw filling capacity on quality and fertilizing ability of buffalo semen Vet Med J Giza 51, 553–566 Sukhato P, Thongsodseang S, Utha A, Songsasen N, 2001: Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa Anim Reprod Sci 67, 69–77 Tahir MN, Bajwa MA, Latif M, Mushtaq M, Shah MH, 1981: Effects of insemination dose and season on conception rates in buffaloes Pak Vet J 1, 161–163 Talevi R, Pelosi S, Sansone G, Graso F, Matasino D, 1994: Effect of different prefreezing rates on buffalo sperm motility and ultrastructure preservation In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong Sao Paulo, Brazil International Buffalo Federation, Roma, Italy, pp 537–539 Taraphder S, Gupta AK, Raina VS, Tomar SS, 2003: Studies on fertility performance of Murrah buffalo bulls Indian J Anim Health 42, 182–184 Tatham B, 2000: Increasing Buffalo Production; Using Reproduction Technology Report Rur Indust Res Corp Dev., Kingston, ACT, Australia Thibier M, Guerin B, 2000: Hygienic aspects of storage and use of semen for artificial insemination Anim Reprod Sci 62, 233–251 Tomar NS, Singh B, 1970: Grading of semen for better fertility rates Indian Vet J 47, 409–413 Tosic J, Walton A, 1950: Metabolism of spermatozoa The formation and elimination of hydrogen peroxide by spermatozoa and effects on motility and survival Biochem J 47, 199–212 Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag Factors Affecting Cryopreservation of Buffalo Spermatozoa Trimeche A, Anton M, Renard P, Gandemer G, Tainturier D, 1997: Quail egg yolk: a novel cryoprotectant for the freeze preservation of Poitou jackass sperm Cryobiology 34, 385– 393 Tuli RK, Mehar S, 1983: Seasonal variation in freezability of buffalo semen Theriogenology 20, 321–324 Tuli RK, Singh M, Matharoo JS, 1981a: Fertility trial under field conditions with frozen buffalo bull semen using Tris yolk glycerol extender Indian J Dairy Sci 34, 456–458 Tuli RK, Mehar S, Matharoo JS, 1981b: Effect of different equilibration times and extenders on deep freezing of buffalo semen Theriogenology 16, 99–104 Valdez CA, Bautista JAN, Rio ASD, Flor JACG, Torres EB, 2003: Ethylene glycol as a cryoprotectant for freezing buffalo spermatozoa: I Effects of concentration and equilibration time on post-thaw survival Philipp J Vet Med 40, 37–43 Vale WG, 1997: Sperm cryopreservation Bubalus Bubalis 1, 129–140 Wales RG, White IG, 1959: The susceptibility of spermatozoa to temperature shock J Endocrinol 19, 211–220 Watson P, 1976: The protection of ram and bull spermatozoa by low density lipoprotein fraction of egg yolk during storage at 5°C and deep freezing J Therm Biol 1, 137–141 Watson PF, 1979: The preservation of semen in mammals In: Finn CA (ed.), Oxford Reviews of Reproductive Biology, 1st edn Oxford Univ Press, Oxford, pp 283–350 Watson PF, 2000: The causes of reduced fertility with cryopreserved semen Anim Reprod Sci 60 61, 481–482 Watson PF, Kunze E, Cramer P, Hammerstedt RH, 1992: A comparison of critical osmolality and hydraulic conductivity 569 and its activation energy in fowl and bull spermatozoa J Androl 13, 131–138 White IG, 1993: Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review Reprod Fertil Dev 5, 639–658 Woelders H, 1997: Fundamentals and recent development in cryopreservation of bull and boar semen Vet Quart 19, 135– 138 Wolff H, Panhans A, Stolz W, Meurer M, 1993: Adherence of Escherichia coli to sperm: a mannose mediated phenomenon leading to agglutination of sperm and E coli Fertil Steril 60, 154–158 Younis M, Samad HA, Ahmad N, Ali CS, 1998: Effect of age and breeding season on the freezability of buffalo bull semen Pak Vet J 18, 219–223 Younis M, Samad HA, Ahmad N, Ahmad I, 1999: Fertility of frozen-thawed semen collected from young, adult and old buffalo bulls during low and peak breeding seasons Pak Vet J 19, 78–80 Yu ZW, Quinn PJ, 1994: Dimethyl sulphoxide: a review of its applications in cell biology Biosci Rep 14, 259–281 Submitted: 04 Jun 2008 Author’s address (for correspondence): Dr SMH Andrabi, Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad 45500, Pakistan E-mail: andrabi123@yahoo.com Ó 2008 The Author Journal compilation Ó 2008 Blackwell Verlag