Factors A Ecting The Quality of Cryopreserved Bu Alo (Bubalus Bubalis) Bull

Reprod Dom Anim 44, 552569 (2009); doi: 10.1111/j.1439-0531.2008.01240.
x ISSN 0936-6768
Review Article Factors Aecting the Quality of Cryopreserved Bualo (Bubalus bubalis) Bull Spermatozoa
SMH Andrabi
Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan
Contents
Storage of bualo (Bubalus bubalis) bull semen in the cryopreserved state is discussed in this article. Fertility rate in bualo following articial insemination with frozenthawed semen is reviewed. To better understand the freezability of bubaline spermatozoa, the available data on biochemical components and the activity of specic enzymes of semen spermatozoa are given. Moreover, the major factors that may inuence the post-thaw viability and fertility of bualo spermatozoa are examined in detail. In addition, suggestions for improvement in cryogenic procedures for bualo spermatozoa are also given.
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 ()196C) state and reviews the major factors aecting the viability and fertility of cryopreserved bualo 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, coolingfreezing and during the thawing procedures (Watson et al. 1992; Holt 2000a,b). Damage occurring during the freezingthawing procedures aect mainly cellular membranes (plasma and mitochondrial) and in the worst case, the nucleus (Blesbois 2007). This damage to membranes has consequences on viability and dierent metabolic factors including adenosine triphosphate (ATP) concentration in spermatozoa. Therefore, such changes in the integrity of spermatozoa aect the viability and fertility. Table 1 summarizes dierent stresses encountered by the cell and the eect on the cell of each stressor during the cryogenic processes. The rst successful freezing of bualo semen was reported by Roy et al. (1956). Basirov (1964) was the rst to report the pregnancy with frozenthawed bualo bull spermatozoa. Since then, AI has been adopted in bualoes; however, it remains unpopular because of poor fertility rate with frozenthawed 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 bualo spermatozoa with AI is presented in Table 2. A critical assessment in term of rst service conception rate of the reports given in Table 2 is dicult, 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 bualo with AI using frozenthawed semen is not comparable with that of cattle.
Introduction
The domestic bualo, Bubalus bubalis, is a distinct species within the Bovidae family. The bualo 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 bualoes play a prominent role in rural livestock production providing the milk, meat and work draft force. In recent decades, bualo farming has also expanded widely in Mediterranean areas and in Latin America. Only in India and Pakistan are there well-dened bualo breeds (Drost 2007). There are 18 river bualo breeds in South Asia, which are further classied into ve major groups designated as the Murrah, Gujarat, Uttar Pradesh, Central Indian and South Indian breeds. The Nili-Ravi bualo, belonging to the Murrah group, is recognized as the highest milk-producing breeds of bualo (Cockrill 1974). The swamp bualo 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., articial insemination (AI). Moreover, the quality of frozenthawed semen is one of the most inuential factors aecting the likelihood of conception (Saacke 1984). Application of AI with frozen thawed semen has been reported on a limited scale in bualo, because of poor freezability and fertility of bualo 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
2008 The Author. Journal compilation 2008 Blackwell Verlag
Factors Aecting Cryopreservation of Bualo Spermatozoa

Table 1. 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 eects on membranes, including solubilization of membrane proteins Destabilization of the lipid bilayers Unknown Mechanical damage to membranes and the cytoskeleton Possible limitation of diusion processes Denaturation of proteins Membrane damage
553
Tables 3 and 4. The values of dierent constituents given in Tables 3 and 4 show that bualo 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 dened extenders and cryogenic procedures that are species specic, and may result in the improvement of viability and fertility of frozenthawed bualo spermatozoa. Buffer Dilution of semen in a suitable buer is one of the important factors aecting sperm survival during cryopreservation (Rasul et al. 2000). An ideal buer should have: (i) pH between 6 and 8, preferably 7; (ii) maximum water solubility and minimum solubility in all other solvents; (iii) minimum salt eects; (iv) minimum buer concentration; (v) least temperature eect; (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 buering system for the cryopreservation of bualo spermatozoa has been in progress for sometime (Rasul et al. 2000). Several studies have concentrated on the use of chemically dened buers for bualo semen. In this regard, Matharoo and Singh (1980) tested citrate, Tris or citric acid as buers for deep-freezing of bualo 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 buer. Ahmad et al. (1986) found that Triscitric acid based extender is suitable for the cryopreservation of bualo spermatozoa in terms of post-thaw motility and survivability. Later on, Dhami and Kodagali (1990) studied the eects of semen extenders based on Tris or citrate buer. It was reported that Tris-based extender improved the freezability of bualo 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 bualo spermatozoa. They found that with Tris-based extender there was least release of lactic dehydrogenase and sorbitol dehydrogenase in bualo spermatozoa during cryopreservation followed by citrate and citric acid-based extenders. In addition, Tris provided the highest protection against acrosomal damage compared to other buers tested. Dhami et al. (1994) studied the eects 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-buer with Laiciphos (IMV, LAigle, France; containing laiciphos, egg yolk, distilled water and unknown buer) and Biociphos (IMV, France; containing biociphos with
Conception rate in bualoes inseminated with frozen thawed semen under eld condition is approximately 30% (Chohan et al. 1992; Anzar et al. 2003). Published reliable studies on the fertility of liquid stored bualo 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 bualoes (Tomar and Singh 1970; Akhter et al. 2007). Therefore, a pregnancy rate higher than 50% is regarded as a good result after AI with frozenthawed spermatozoa in bualo (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 frozenthawed spermatozoa. From above, it is suggested that cryopreservation adversely aects the viability and the fertilizing potential of bualo bull spermatozoa. Therefore, there is a need to discuss in depth the major factors inuencing the successful cryopreservation of bualo spermatozoa.
Factors Affecting Freezability

Biochemical characteristics of semen It is reported that bualo 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 specic biochemical factors that aect the ability of spermatozoa to prevent damages caused by the cryogenic procedures. One of the many possible causes of lower freezability of bualo bull semen compared to cattle bull can be due to the dierences 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 bualo 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 bualo 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 specic enzymes of semen are given in
554
Table 2. Fertility rate in bualo following AI with frozenthawed 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 Extender Skim milk powderyolkglycinecitrate fructoseglycerol Yolkcitrateglycerol Yolkcitrateglycerol, Citric acidwheyglycerol and Trisyolkglycerol Trisyolkglycerol Trisyolkglycerol Trisyolkglycerol Information not provided Lactosefructoseyolkglycerol, Skim milkfructoseyolkglycerol and Trisfructoseyolkglycerol Information not available Yolkglycerol with milk or lactose or fructose and lactose Yolklactosefructoseglycerol Trisfructoseyolkglycerol Trisfructoseyolkglycerol with or without additives (L-cysteine HCl H2O, sheep hyaluronidase, beta-amylase or acetylcholine chloride) Information not provided Trisegg yolkglycerol Trisfructoseyolkglycerol, Yolkcitrateglycerol and Lactoseyolk glycerol Information not provided Information not provided Trisyolkglycerol, Citrateyolkglycerol and Lactoseyolkglycerol, with or without (control) cysteine, EDTA and ranose TrisTES and Trisyolk Triscitric acidfructoseyolkglycerol and Whole cows milkyolkglycerol Lactosefructoseyolkglycerol Information not available Information not provided Trisfructoseyolkglycerol 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 Triscitric acidfructoseyolkglycerol Total number of rst AI 109 Information not available 315 72 159 825 61 952 3220
SMH Andrabi
Over all rst service aCR (%) 45.0 40.6 53.93 45.8 35.22 39.30 51.78 37.4
Singh and Singh 1988 Ahmad et al. 1988 Bhavsar et al. 1988 Bhavsar et al. 1989a Bhavsar et al. 1989b
218 2745 1908 Information not available 3791
41.0 44.7 39.2 45.85 46.0
Singh 1990 Haranath et al. 1990 Dhami and Kodagali 1990
Information not provided Information not available 3412
39.7 51.53 39.9
Dhami and Kodagali 1991 Hassan and Zia Ur 1994 Dhami et al. 1994
2995 1110 853
40.1 65.26 57.95
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
a
Information not available 806 971 217 6762 178 202 1941 Information not available Information not available 67 Information not available Information not available 432
53.14 63.98 41.8 42.5 52.0 37.0 34.95 59.15 40.75 50.6 48.0 29.87 29.0 53.0
Pregnancies were conrmed through rectal palpations. Bualo were synchronized with a progesterone-releasing intravagiral device (PRID) containing progesterone and oestradiol benzoate, for 10 days. Seven days after insertion of PRID the bualo 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-B) into the vagina. Each bualo was injected intramuscularly with 1 mg of oestradiol benzoate (CIDIROL) 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 bualoes bearing a functional corpus luteum.
b
glycerol, egg yolk, distilled water and unknown buer) for cryopreservation of bualo 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 buer for cryopreservation of bualo semen. The buers tested were tri-sodium citrate, Triscitric acid, TrisTes or TrisHepes. They found that Tris citrate tended to be better in term of improving the postthaw motion characteristics of bualo spermatozoa. Nonetheless, plasma membrane integrity and normal acrosomes of spermatozoa did not vary because of
buering 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 buers particularly, Triscitric acid may provide the most satisfactory buering system to improve the post-thaw freezability and consequently may also improve the fertility of bualo spermatozoa. It is believed that zwitterion buers have pH nearer to the pKa (acid dissociation constant). Also there pKa is least inuenced by temperature as compared to other buers (Graham et al. 1972).

Table 3. Biochemical composition of bualo semen
Characteristic of component Lactic acid Reference Rattan et al. 1980 Whole semen Seminal plasma 23.47 mg 100 ml Spermatozoa Comment
555
Dabas et al. 1984
82 6 mg 100 ml
167 9 lg 1011 cells 0.066 0.014 lmol 109cells
Ascorbic acid
Jain 1987 Banerjee and Ganguli 1973
0.091 0.011 lmol ml 6.2 0.8 mg 100 ml
0.024 0.003 lmol ml 3.9 0.5 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
368.12 430.92 mg 100 ml 623.8 83.6 mg 100 ml 815.71 mg 100 ml
Amount of lactic acid in cattle bull seminal plasma is 72 5 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 519.07618.93 mg 100 ml (Salem and Osman 1972)
Banerjee and Ganguli 1973 Rattan et al. 1980
Lipids
Jain and Anand 1976
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
91.84 3.91 141.88 3.12 mg 100 ml 0.594 mg ml 0.548 mg 109 cells
Phospholipids
Jain and Anand 1976
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 104412 mg 100 ml (RoyChoudhury 1970) Amount of phospholipids in seminal plasma of cattle bull is 1.491 mg ml (Jain and Anand 1976)
Sidhu and Guraya 1979 Sarmah et al. 1983
0.1735 0.0256 mg ml 0.069 0.02 mg ml 21.7 1.0% of total phospholipids
0.3074 0.0923 mg 109 cells 0.064 0.02 mg 109 cells 30.4 1.4% of total phospholipids
Phosphatidyl choline
Jain and Anand 1976
Sarmah et al. 1983
34.1 1.8% of total phospholipids
Phosphatidal choline (choline plasmogen)
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
Table 3. Continued
Characteristic of component Phosphatidyl ethanolamine Reference Jain and Anand 1976 Whole semen Seminal plasma 11.7 1.5% of total phospholipids Spermatozoa 10.8 2.0% of total phospholipids
SMH Andrabi
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 dier 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 dier 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
Sarmah et al. 1983
Phosphatidal ethanolamine (ethanolamine plasmogen)
Jain and Anand 1976
Sarmah et al. 1983
Sphinogomyelin
Jain and Anand 1976
Sarmah et al. 1983
Phosphatidyl serine
Jain and Anand 1976
Phosphatidyl serine + phosphatidyl inositol
Sarmah et al. 1983
Phosphatidyl inositol
Jain and Anand 1976
Lysophosphatidyl choline
Jain and Anand 1976
Sarmah et al. 1983

Table 3. Continued
Characteristic of component Lysophosphatidyl ethanolamine Reference Jain and Anand 1976 Whole semen Seminal plasma 5.6 1.5% of total phospholipids Spermatozoa 4.4 1.0% of total phospholipids Comment
557
Sarmah et al. 1983
Lysophosphatidyl serine
Jain and Anand 1976
Diphosphatidyl glycerol (cardiolipin)
Jain and Anand 1976
Sarmah et al. 1983
Phosphatidic acid
Jain and Anand 1976
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
32.49 5.10 lmol ml
Aspartic acid
Chaudhary and Gangwar 1977 Chaudhary and Gangwar 1977 Chaudhary and Gangwar 1977 Chaudhary and Gangwar 1977 Chaudhary and Gangwar 1977 Chaudhary and Gangwar 1977
0.395 mM
Glutamic acid
4.28 mM
Serine
0.60 mM
Alanine
0.413 mM
Glycine
1.34 mM
Lysine
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)
558
Table 3. Continued
Characteristic of component Deoxyribo-nuclease Reference Chauhan et al. 1975 Whole semen Seminal plasma Spermatozoa 2007.33 112.01 KU ml
SMH Andrabi
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 2 BU 1011 cell respectively (Dabas et al. 1984) Amount of alkaline phosphatase in seminal plasma and spermatozoa of cattle bull is 246 8 BU 100 ml and 54 3 BU 1011 cell, respectively (Dabas et al. 1984)
Acid phosphatase
Chauhan and Srivastava 1973 Dabas et al. 1984 Chauhan and Srivastava 1973 Dabas et al. 1984
Alkaline phosphatase
315.31 22.66 BU 100 ml 194 10 BU 100 ml 312.50 24.04 BU 100 ml 270 9 BU 100 ml
39 6 BU 1011 cell
63 6 BU 1011 cell
Values are mean SEM.
Table 4. Phospholipid composition (% of total phospholipids) of plasma membrane of bualo and cattle bull spermatozoa
Phospholipid Phosphatidyl ethanolamine Phosphatidyl choline Phosphatidyl serine Phosphatidyl inositol Sphinogomyelin Lysophosphatidyl choline Phosphatidyl glycerol Diphosphatidyl glycerol
a
Bualo (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
Values are mean SE.
Additionally, the dierences regarding ecacy of dierent buers suggest that bualo spermatozoa are more prone to freezing stress as compared to cattle bull spermatozoa possibly because of biochemical factors that inuence membrane uidity during cryogenic preservation (refer to Table 4). Therefore, there is a need to study the inuence of selected buers 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 15 mol l, depending on cell type and conditions of exposure (Fuller and Paynter 2004). More specically, 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 bualo semen, several studies have been carried out in an attempt to nd the optimum levels of glycerol and glycerolization. In this context, Jainudeen and Das (1982) studied the eect of two glycerolization procedures (one step vs two steps) and
the inuence of glycerol level in the extender (3%, 5% or 7%). They found that glycerolization procedure had no signicant eect on sperm survival traits like motility and acrosomal integrity. They also found that post-thaw motility of spermatozoa was signicantly 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 bualo spermatozoa. Ramakrishnan and Ari (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 eects of dierent 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 signicantly 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 conrmed that single step is more suitable for the cryopreservation of bualo spermatozoa in terms of post-thaw forward motility. Ethylene glycol could be another option for the cryopreservation of bualo spermatozoa. Permeability of ethylene glycol was found to be higher than glycerol
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in spermatozoa of dierent 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 aect the viability of frozen bualo spermatozoa with ethylene glycol as a cryoprotectant. Further studies, are also suggested for testing propylene glycol as a cryoprotectant for bualo spermatozoa. Dimethyl sulfoxide (DMSO) is a rapid penetrating cryoprotectants having lower molecular weight than glycerol. Also DMSO may inhibit harmful eect 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 37C or at 4C as a cryoprotectant for bualo spermatozoa. The concentrations (%) of glycerol and DMSO adjusted were 0 : 0, 0 : 1.5, 0 : 3; 3 : 0, 3 : 1.5, 3 : 3; and 6 : 0, 6 : 1.5, 6 : 3 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 37C, provided the maximum cryoprotection to the motility apparatus, and plasma membrane integrity of bualo spermatozoa in Tris citric acid based extender. The exact mechanism involved in the antagonist eect of DMSO on the cryoprotection ability of glycerol is not understood. Moreover, the lethal eect of DMSO is attributed to its toxic eect 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 57% added initially in the extender may be suitable for the cryopreservation of bualo bull spermatozoa. On the other hand, development of less toxic cryoprotectant could make a signicant contribution in improving the quality of frozenthawed bualo spermatozoa. Non-permeable cryoprotectant Egg yolk is a common component of semen freezing extenders for most of the livestock species, including the bualo (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-
brane. A second hypothesis suggests that phospholipids present in LDL protect sperm by forming a protective lm 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 freezethaw 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 bualo 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 eects 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 dierent levels of egg yolk in extender as a nonpermeable cryoprotectant for bualo 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 eect of dierent levels of egg yolk (0%, 1%, 5%, 10% and 20%) in Tris-based extender on sperm motility and survival before and after freezing in bualo. They found that the best post-thaw motility and survivability was with 5% yolk. Singh et al. (1999) studied the eect of dierent levels of egg yolk on freezability of bualo 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 bualo 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 bualo 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 bualo 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 dierent avian species in freezing extender is attributed to the dierences in biochemical composition of the yolks (Trimeche et al. 1997; Bathgate et al. 2006). Studies investigating the inuence of egg yolk from dierent 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 aorded to the sperm
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(Trimeche et al. 1997). This is of interest to mention that Bathgate et al. (2006) reported a signicant dierence 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 bualo spermatozoa in the study by Andrabi et al. (2008). It is, also proposed that supplementation of cryodiluent with quail egg yolk for bualo 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 ndings 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 bualo 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 eect 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 bualo spermatozoa. They concluded that PEG 20 added to extender preserved the lipids of frozen bualo spermatozoa. Further studies are required to nd out that PEG 20 may be a better option for the cryopreservation of bualo spermatozoa. Sugars that are not capable of diusing across a plasma membrane, such as lactose, sucrose, ranose, 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 bualo 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 bualo 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 eect of 1% ranose in semen diluents (Trisfructoseyolkglycerol, egg yolkcitrateglycerol or lactoseegg yolkglycerol) on enzyme leakage (lactate dehydrogenase) from bualo spermatozoa during freezing. They found that the postthaw quality of spermatozoa was better with ranose 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 bualo spermatozoa. Antibiotic It is documented that bacteria in semen and their control via addition of antibiotics in freezing diluents may aect the viability or fertility of cryopreserved bovine spermatozoa (Thibier and Guerin 2000; Morrell 2006). Presence of bacteria in the ejaculates can aect fertilization directly (Morrell 2006), by adhering to spermatozoa (Bolton et al. 1986; Wol 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 eect by producing toxins (Morrell 2006). Thus, in the use of AI, it is important to control eciently 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 bualo bull semen (Sansone et al. 2000; Akhter et al. 2008). Regarding control of bacteriospermia in bualo bull semen with streptomycin and penicillin (SP), it was found that it is not an eective 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 bualo 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 noroxacin (200 lg ml) are the antibiotics of choice to be added in extender for ecient preservation of bualo spermatozoa. Recently, Hasan et al. (2001) and Akhter et al. (2008) investigated the eects 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 bualo bull semen as judged by total aerobic bacterial count and or in vitro antibiotic sensitivity. Moreover, GTLS is not detrimental to spermatozoal viability of bualo bull. It is relevant to mention that Andrabi et al. (2001) have reported a better conception rate with frozenthawed semen having GTLS compared to SP (55.2% vs 41.66%). It is therefore, suggested that GTLS in extender is more ecient for the preservation of bualo spermatozoa. Further, that testing of wider range of new antibiotic is recommended in cryodiluents for improvement in quality of frozenthawed bualo spermatozoa.
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Other additives Keeping in view the poor freezability of bubaline semen attempts have been made to improve the basic buers developed to minimize the deleterious eects 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 bualo spermatozoa. In this regard, Bhosrekar et al. (1990) studied the eect of addition of caeine or triethanolamine lauryl sulphate to Triscitric acid-based extender. They reported that the addition of the detergent improved the post-thaw spermatozoa motility. However, inclusion of caeine to extender did not made any improvement in motility. It is believed that the protective eect 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 caeine to make any improvement is not understood. Dhami and Sahni (1993) studied the eect of 0.1% cysteine or 0.1% EDTA (sperm membrane stabilizer and capacitation inhibitor) in semen diluents (Tris fructoseyolkglycerol, egg yolkcitrateglycerol or lactoseegg yolkglycerol) on enzyme leakage (lactate dehydrogenase) from bualo 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 eect of addition of ascorbic acid in the diluent on the quality of deep frozen bualo bull semen. They found that inclusion of ascorbic acid (2.5 mM) in the semen diluent yielded a signicantly higher post-thaw motility and survivability. The antioxidant eect 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 eect of vitamin A, D and E in extender on motility, survivability and acrosomal integrity of cryopreserved bualo bull spermatozoa. It was suggested that vitamin E at 0.3 mg ml exhibited the best eects. 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 bualo spermatozoa, addition of sodium pyruvate (1.25 mM) to the extender resulted in signicantly better post-thaw progressive motility and viability. The benecial eect of pyruvate and a-ketoacids is attributed to its antioxidant property. Shukla and Misra (2005) studied dierent 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 signicantly
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 eects of addition of oviductal proteins obtained from various stages of the oestrous cycle to Tris-based extenders on spermatozoa characteristics in bualoes. They found that oviductal proteins dierentially aected 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 bualo spermatozoa during cryopreservation. The benecial actions conveyed by oviductal uid are presently unknown; however, the identication of catalase in cow oviductal uid by Lapointe et al. (1998) suggests that it may be a mechanism by which the oviductal uid reduces the damage caused by reactive oxygen species to the spermatozoa. Recently, Shukla and Misra (2007) conducted a study to improve bualo 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 frozenthawed 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 eects in terms of improvement in the quality of frozen thawed bualo 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 benecial eects on cryopreservation of bualo 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 frozenthawed semen (Salamon and Maxwell 2000). In farm animals, the semen has been diluted with specic volumes of extenders or by diluting semen to a specic spermatozoa concentration. Dilution rates of
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1 : 1 to 1 : 12 have been successfully used for bualo semen. Perhaps a better of diluting semen, for comparison purposes, is based on the sperm concentration (Purdy 2006). Reports of bualo 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 4C or 5C. 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 5C, and do not happen below 0C (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 buering system (Marshall 1984). It has been empirically determined that cooling cattle bull spermatozoa from body temperature to 5C performed at a rate of 10C h has minimum deleterious eects (Parks 1997). In this regard, Dhami et al. (1992) studied the eect of cooling rates (5, 30, 60 and 120 min from 10 to 5C vs 120 min from 28 to 5C) on the deep freezing of bualo semen diluted in Tris-based extender. Their results suggest that bualo semen can be frozen successfully after 30 min of cooling at 10C 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 (buer and cryoprotectant) used and could easily interact with other cryogenic procedures (Marshall 1984). In this regard, Tuli et al. (1981b) examined equilibration of bualo semen diluted in Tris or citric acid-based extender for 2, 4 or 6 h. They found that post-thaw sperm survivability was better after 4 h equilibration than after 2 or 6 h. Talevi et al. (1994) cooled bualo semen from 28C to 5C in 15 min and then equilibrated at 5C for 1 h 45 min (fast cooling) or cooled it in 1 h and equilibrated for 1 h (slow cooling). They found that post-thaw sperm motility was signicantly higher using the slow than the rapid cooling method. Conversely, the rate of cooling had no signicant eect on acrosome integrity. Dhami et al. (1996) conducted a study to determine the relative ecacy of four cooling rates (10 30C to 5C; 1 and 2 h each) and two equilibration periods at 5C (0 and 2 h) for cryopreservation of bualo ejaculates. They concluded that slow cooling of straws from 30 to 5C for 2 h compared with faster cooling (1 h) or lower initial
temperature (10C) and 2 h of equilibration at 5C appeared necessary for successful cryopreservation of bualo 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 )50C) 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 (50100C) 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 40C is practiced in general for cryopreservation during the critical temperature zone (Anzar et al. 2002). Sukhato et al. (2001) determined the eects of freezing rate and intermediate plunge temperature (cooling at 10, 20 or 30C min each to )40, )80 or )120C before being plunged into liquid nitrogen) on post-thaw quality and fertility of bualo spermatozoa. They found that cooling freezing spermatozoa from 4 to )120C, either at 20 or 30C min 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 bualo bull semen. It was concluded that freezing at the rate of 17.32C min between +4C and )40C with programmable freezer produced better quality frozen semen than the conventional method of freezing. More recently, Rasul (2000) examined the eect of freezing rates on post-thaw viability of bualo spermatozoa extended in Triscitric acid-based extender. The freezing rates examined between 4 and )15C were 3 or 10C min, whereas the freezing rates investigated between )15 and )80C were 10, 20 or 30C min. It was concluded that the dierent 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 freezethaw procedure, the warming phase is just as important to the survival of spermatozoa as the freezing phase. Spermatozoa that have survived cooling to )196C still face the challenge of warming and thawing, and thus must traverse twice the critical temperature zone i.e., from )5 to )50C (Marshall 1984). The thawing eect depends on whether the rate of cooling has been suciently high to induce intracellular freezing, or low enough to produce cell dehydration. In the former case, fast thawing is required to prevent
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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 uctuation and subsequently protein denaturation and cell death. A practical thaw for cattle bull spermatozoa, recommended by most AI organizations, is as 35C water bath for at least 30 s (Marshall 1984). For cryopreservation of bualo spermatozoa in Tris-based extender, Rao et al. (1986) tested two thawing rates (37C for 30 s and 75C for 9 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 eect of thawing rates (40C for 60 s, 60C for 15 s and 80C for 5 s) on post-thaw motility of bualo spermatozoa cryopreserved in Tris-based extender. They reported that thawing at 60C for 15 s yielded a higher sperm motility compared to other rates. In another study, Dhami et al. (1996) determined the thawing rates for bualo semen. The thawing rates investigated were 4C for 5 min, 40C for 1 min or 60C for 15 s. They concluded that thawing of semen at 60C for 15 s yielded high post-thawing spermatozoal recovery and longevity. Sukhato et al. (2001) determined the eect of thawing rates on motility and acrosome integrity of bualo spermatozoa. Thawing of spermatozoa was performed at the rate of (rapid) 1000C or (slow) 200C min. They concluded that rapid thawing was superior to slow warming. The above-mentioned studies demonstrate that an eective cryopreservation procedure for bualo spermatozoa can be derived by the systematic examination of various cryobiological factors. Therefore, from these studies the cryogenic procedures for bualo semen can be outlined as; cooling from 37 or 39 to 4C at the rate of 0.20.4C min, equilibration, at least 2 h at 4C, freezing of straws approximately 4 cm above liquid nitrogen for 1020 min, or by the fast freezing rates (programmable freezing), and thawing at 4560C for at least 15 s. However, there is still need to improve the processing techniques for cryopreservation of bualo spermatozoa. It is suggested that to devise ecient cooling freezing rates for bualo 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 bualo semen can also be aected by the season of collection i.e., by environmental factors like temperature, humidity and day length in a particular season. For the rst time Tuli and Mehar (1983) studied the seasonal variation in freezability of bualo semen diluted in Tris-based extender. They found that post-thaw spermatozoa motility, signicantly increased in winter than summer season. After that, Heuer et al. (1987) studied the eect of season on in vivo fertility of frozen bualo semen diluted in chemically dened buers. They
reported that semen collected in November (winter) produced signicantly 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 bualo fertility to the male. Bhavsar et al. (1989a) also studied the monthly variations in freezability and fertility of bualo 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 signicantly 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 bualo bull semen. They found from the data of over a 4-year period that the season signicantly aected 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 bualo bull semen. They found that post-thaw semen motility was signicantly aected by season of collection, being lowest in summer and highest in winter (DecemberJanuary). Younis et al. (1998) studied the freezability of semen collected during the low breeding season (MayJuly) and the peak breeding season (SeptemberNovember) in young (34 years), adult (68 years) and old (1215 years) bualo 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 frozenthawed semen were signicantly higher during the low breeding season than in the peak breeding season. Recently, Koonjaenak et al. (2007a) studied the seasonal eect on quality of frozenthawed bualo spermatozoa diluted in Tris-based buer. They compared post-thaw sperm quality over three seasons of the year (rainy: JulyOctober; winter: NovemberFebruary; and summer: MarchJune), with distinct ambient temperature and humidity. Their conclusion was that postthaw plasma membrane integrity and stability were signicantly 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 conrming that vitality of bualo spermatozoa remain comparatively poorer during this season. Moreover, it is suggested that to increase fertility rate in bualo, 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 frozenthawed bualo sperm nuclear DNA fragmentation by owcytometry and head morphology over three seasons in tropical Thailand (the rainy season, JulyOctober; winter, NovemberFebruary; and summer, MarchJune). 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 aected by the year of semen collection and processing. The proportion of morphologically
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SMH Andrabi Ahmad K, Chaudhry RA, 1980: Cryopreservation of bualo semen. Vet Rec 106, 199201. Ahmad M, Ahmad KM, Khan A, 1986: Cryopreservation of bualo spermatozoa in Tris (hydroxymethyl-aminomethane). Pak Vet J 6, 13. Ahmad KM, Khan A, Ahmad M, 1988: Fertility of Nili-Ravi bualo bull frozen semen at dierent thawing temperatures. Buffalo J 4, 5155. Ahmad Z, Anzar M, Shahab M, Ahmad N, Andrabi SMH, 2003: Sephadex and sephadex ion-exchange ltration improves the quality and freezability of low-grade bualo semen ejaculates. Theriogenology 59, 11891202. Ahmed K, Greesh M, 2001: Eect of antibiotics on the bacterial load and quality of semen of Murrah bualo bulls during preservation. Indian J Anim Reprod 22, 7980. Ahmed K, Greesh M, Tripathi RP, 2001a: Eect of dierent antibiotics on semen quality during post thaw incubation at 37C. Indian J Anim Reprod 22, 81. Ahmed K, Kumar AA, Mohan G, 2001b: Bacterial ora of preputial washing and semen of Murrah bualo bulls and their antibiotic sensitivity pattern. Ind J Comp Microbiol Immunol Infect Dis 22, 6364. Aisen EG, Medina VH, Venturino A, 2002: Cryopreservation and post-thawed fertility of ram semen frozen in dierent trehalose concentrations. Theriogenology 57, 18011808. Akhter S, Ansari MS, Andrabi SMH, Ullah N, Qayyum M, 2007: Eect of antibiotics in extender on fertility of liquid bualo bull semen. Pak Vet J 27, 1316. Akhter S, Ansari MS, Andrabi SMH, Ullah N, Qayyum M, 2008: Eect of antibiotics in extender on bacterial and spermatozoal quality of cooled bualo (Bubalus bubalis) bull semen. Reprod Domest Anim 43, 272278. Ala Ud D, Chaudhry RA, Ahmad K, 1981: Eect of dierent extenders on freezability of bualo semen. Pak Vet J 1, 5961. Aleem M, Chaudhry RA, Khan NU, Rizvi AR, Ahmed R, 1990: Occurrence of pathogenic bacteria in bualo semen. Buffalo J 6, 9398. Al-Hakim MK, Graham EF, Schmehl ML, 1970: Free amino acids and amino compounds in bovine seminal plasma. J Dairy Sci 53, 8488. Ali H, Din A, Samad HA, Ali S, Sabri MA, 1994: Comparative eects of combiotic, ampicillin and gentamycin sulphate on motility percentage, liveability and absolute index of liveability in the bualo bull semen. Pak Vet J 14, 223227. Amin AS, Darwish GM, Maha SZ, Hassan HM, 1999: Trial to control Chlamydia psittaci in processed bualo-semen. Assi Vet Med J 40, 319332. Andrabi SMH, Ahmad N, Abbas A, Anzar M, 2001: Eect of two dierent antibiotic combinations on fertility of frozen bualo and Sahiwal bull semen. Pak Vet J 21, 166169. Andrabi SMH, Siddique M, Ullah N, Khan LA, 2006: Eect of reducing sperm numbers per insemination dose on fertility of cryopreserved bualo bull semen. Pak Vet J 26, 1719. Andrabi SMH, Ansari MS, Ullah N, Anwar M, Mehmood A, Akhter S, 2008: Duck egg yolk in extender improves the freezability of bualo bull spermatozoa. Anim Reprod Sci 104, 427433. Anzar M, He L, Buhr MM, Kroetsch TG, Pauls KP, 2002: Sperm apoptosis in fresh and cryopreserved bull semen detected by ow cytometry and its relationship with fertility. Biol Reprod 66, 354360. Anzar M, Farooq U, Mirza MA, Shahab M, Ahmad N, 2003: Factors aecting the eciency of articial insemination in cattle and bualo 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,
abnormal sperm head shapes was low, with no signicant dierences between seasons. However, DFI was signicantly related to the proportion of loose abnormal sperm heads. It was concluded that frozenthawed bualo sperm chromatin is not critically damaged by cryopreservation or aected 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 rst 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 modications may explain, in part, the reduced freezability of bualo semen collected during the summer. Few scattered reports are available that describe the dierences in chemical composition of bualo seminal plasma and spermatozoa under dierent climatic conditions (Singh et al. 1969; Mohan et al. 1979; Sidhu and Guraya 1979). However, the information given in these studies are insucient to explain the variation in freezability of bualo spermatozoa during the dierent seasons. Therefore, detailed studies should be carried out to ascertain the biochemical or structural dierences in seminal plasma, spermatozoa, or plasmalemma, which might be inuencing the freezability of bualo spermatozoa during the dierent months seasons.
Conclusions
Viability and fertility of frozenthawed bualo bull spermatozoa is considerably lower than that of cattle bull. Several buers, 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 bualo spermatozoa is necessary according to the specic requirements. Moreover, there is a need to develop biochemically dened extenders and cryogenic procedures that may result in improvement in viability and fertility of frozenthawed bualo spermatozoa. Besides this, the season during which the semen is collected should also be considered as a variable aecting quality of cryopreserved bualo spermatozoa.
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568 Rao AVN, Haranath GB, Sekharam GS, Rao JR, 1986: Eect of thaw rates on motility, survival and acrosomal integrity of bualo spermatozoa frozen in medium French straws. Anim Reprod Sci 12, 123129. Rasul Z, 2000: Cryopreservation of buffalo semen. PhD Thesis, Quaid-i-Azam University, Islamabad, Pakistan. Rasul Z, Anzar M, Jalali S, Ahmad N, 2000: Eect of buering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of bualo spermatozoa. Anim Reprod Sci 59, 3141. Rasul Z, Ahmed N, Anzar M, 2007: Antagonist eect of DMSO on the cryoprotection ability of glycerol during cryopreservation of bualo sperm. Theriogenology. 68, 813819. Rattan PJS, Rao MB, Krishnan CPA, 1980: Biochemical studies on bualo-bull (Bos bubalis) semen. Indian Vet Med J 4, 6570. Rohilla RK, Tuli RK, Goyal RL, 2005: Comparative study of the eects of cryoprotective agents in freezing Murrah bualo bull semen. Indian J Vet Res 14, 3743. Roy A, Srivastava RK, Pandey MD, 1956: Deep freezing of bualo semen diluted and preserved in glycine-egg yolk medium. Indian J Dairy Sci 9, 6162. RoyChoudhury PN, 1970: Total cholesterol content in bull semen. Indian Vet J 47, 146150. de Ruigh L, Bosch JC, Brus MC, Landman B, Merton JS, 2006: Ways to improve the biosecurity of bovine semen. Reprod Domest Anim 41, 268274. Saacke RG, 1984: Semen quality: importance of and inuencing factors. In: Proceedings of 10th NAAB Tech. Conf. A. I. Reprod Milwaukee, WI, USA. National Association of Animal Breeders, Columbia, USA, pp. 3036. Sagdeo LR, Chitnis AB, Kaikini AS, 1991: Eect of seasonal variations on freezability of Surti bualo bull semen. Indian J Anim Reprod 12, 13. 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, 77111. Salem HM, Osman SA, 1972: Seasonal variations in the chemical constituents of blood and semen in bualo and cattle. Alexandria J Agric Res 20, 5155. Sansone G, Nastri MJF, Fabbrocini A, 2000: Storage of bualo (Bubalus bubalis) semen. Anim Reprod Sci 62, 55 76. Sarmah BC, Kaker ML, Razdan MN, 1983: Total lipids and phospholipids in bualo semen (Bubalus bubalis). Theriogenology 20, 521527. Senatore EM, Verberckmoes S, Pascale M, Presicce GA, 2004: A deep utero-tubal semen deposition in Mediterranean Italian bualoes using a new articial insemination device. Reprod Fertil Dev 16, 133. Shannon P, 1972: The eect 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 Articial Insemination, Milan, Italy, pp. 14401442. Shukla MK, Misra AK, 2005: Eect of antioxidants alpha tocopherol, ascorbic acid and n-propyl gallate on Murrah semen cryopreservation. Buffalo J 21, 2738. Shukla MK, Misra AK, 2007: Eect of Bradykinin on Murrah bualo (Bubalus bubalis) semen cryopreservation. Anim Reprod Sci 97, 175179. Sidhu KS, Guraya SS, 1979: Eects of seasons on physiochemical characteristics of bualo (Bubalus bubalis) semen. Indian J Anim Sci 49, 884889. Singh B, 1990: Sampling methodology for the study of eectiveness of articial insemination in bualoes under eld conditions. Indian J Anim Sci 60, 773776.
SMH Andrabi Singh M, Pant HC, 2000: Eect of post-thaw incubation on semen quality of bualo bulls comparison with cattle. Buffalo Bull 19, 5154. Singh B, Singh D, 1988: Factors aecting conception rate in bualo through articial insemination under eld conditions. Indian J Anim Sci 58, 798799. Singh B, Mahapatro BB, Sadhu DP, 1969: Chemical composition of cattle and bualo spermatozoa and seminal plasma under dierent climatic conditions. J Reprod Fert 20, 175 178. Singh M, Matharoo JS, Chauhan FS, 1980: Preliminary fertility results with frozen bualo bull semen using Tris extender. Theriogenology 13, 191194. 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 bualo semen in certain extenders. Theriogenology 34, 371378. Singh J, Pangawkar GR, Biswas RK, Kumar N, 1991: Studies on bualo sperm morphology during various stages of freezing in certain extenders. Indian J Anim Reprod 12, 126129. Singh B, Chand D, Singh P, Yadav NK, 1996: Eect of vitamin C addition in the diluent on the quality of deep frozen Murrah bualo bull (Bubalus bubalis) semen. Int J Anim Sci 11, 131132. Singh P, Singh S, Hooda OK, 1999: Eect of dierent level of egg yolk on freezability of bualo semen. Haryana Vet 38, 2628. Singh P, Jindal JK, Singh S, Hooda OK, 2000: Freezability of bualo bull semen using dierent extenders. Indian J Anim Reprod 21, 4142. Singh P, Singh I, Singh S, Sharma RK, 2006: Initial stage glycerolization prevents the incidence of backward sperm motility during cryopreservation and increases bualo semen freezability. Indian J Anim Sci 76, 777779. Sosa GA, El-Deeb ED, El-Sabagh KM, 2003: Interactions of diluents, cryoprotective agents and straw lling capacity on quality and fertilizing ability of bualo semen. Vet Med J Giza 51, 553566. Sukhato P, Thongsodseang S, Utha A, Songsasen N, 2001: Eects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved bualo spermatozoa. Anim Reprod Sci 67, 6977. Tahir MN, Bajwa MA, Latif M, Mushtaq M, Shah MH, 1981: Eects of insemination dose and season on conception rates in bualoes. Pak Vet J 1, 161163. Talevi R, Pelosi S, Sansone G, Graso F, Matasino D, 1994: Eect of dierent prefreezing rates on bualo sperm motility and ultrastructure preservation. In: Vale WG, Barnabe VH, Mattos JCA de, Proceedings of 4th World Buffalo Cong Sao Paulo, Brazil. International Bualo Federation, Roma, Italy, pp. 537539. Taraphder S, Gupta AK, Raina VS, Tomar SS, 2003: Studies on fertility performance of Murrah bualo bulls. Indian J Anim Health 42, 182184. 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 articial insemination. Anim Reprod Sci 62, 233251. Tomar NS, Singh B, 1970: Grading of semen for better fertility rates. Indian Vet J 47, 409413. Tosic J, Walton A, 1950: Metabolism of spermatozoa. The formation and elimination of hydrogen peroxide by spermatozoa and eects on motility and survival. Biochem J 47, 199212.
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Submitted: 04 Jun 2008 Authors address (for correspondence): Dr SMH Andrabi, Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Centre, Islamabad 45500, Pakistan. E-mail: andrabi123@yahoo.com

Factors A Ecting The Quality of Cryopreserved Bu Alo (Bubalus Bubalis) Bull

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Factors A Ecting The Quality of Cryopreserved Bu Alo (Bubalus Bubalis) Bull

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Reprod Dom Anim 44, 552569 (2009); doi: 10.1111/j.1439-0531.2008.01240.

2008 The Author. Journal compilation 2008 Blackwell Verlag

Factors Aecting Cryopreservation of Bualo Spermatozoa

Factors Affecting Freezability

2008 The Author. Journal compilation 2008 Blackwell Verlag

218 2745 1908 Information not available 3791

41.0 44.7 39.2 45.85 46.0

Singh 1990 Haranath et al. 1990 Dhami and Kodagali 1990

Information not provided Information not available 3412

39.7 51.53 39.9

2995 1110 853

40.1 65.26 57.95

2008 The Author. Journal compilation 2008 Blackwell Verlag

Factors Aecting Cryopreservation of Bualo Spermatozoa

Dabas et al. 1984

167 9 lg 1011 cells 0.066 0.014 lmol 109cells

Jain 1987 Banerjee and Ganguli 1973

0.091 0.011 lmol ml 6.2 0.8 mg 100 ml

0.024 0.003 lmol ml 3.9 0.5 mg 100 ml

Banerjee and Ganguli 1973

441.8 31.9 mg 100 ml

444.9 17.4 mg 100 ml

Salem and Osman 1972

368.12 430.92 mg 100 ml 623.8 83.6 mg 100 ml 815.71 mg 100 ml

Banerjee and Ganguli 1973 Rattan et al. 1980

Jain and Anand 1976

1.147 mg 109 cells

Sarmah et al. 1983

1.320 0.030 mg 109 cells

Mohan et al. 1979

91.84 3.91 141.88 3.12 mg 100 ml 0.594 mg ml 0.548 mg 109 cells

Jain and Anand 1976

Sidhu and Guraya 1979 Sarmah et al. 1983

0.1735 0.0256 mg ml 0.069 0.02 mg ml 21.7 1.0% of total phospholipids

Jain and Anand 1976

Sarmah et al. 1983

34.1 1.8% of total phospholipids

28.0 1.2% of total phospholipids

Phosphatidal choline (choline plasmogen)

Jain and Anand 1976

17.3 0.9% of total phospholipids

19.4 1.7% of total phospholipids

2008 The Author. Journal compilation 2008 Blackwell Verlag

Sarmah et al. 1983

10.8 1.4% of total phospholipids

9.3 1.2% of total phospholipids

Phosphatidal ethanolamine (ethanolamine plasmogen)

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

Jain and Anand 1976

2.8 0.4% of total phospholipids

1.5 0.3% of total phospholipids