Biochemical Profiles in Freezable and Non-Freezable Ejaculates of Mithun ( Bos frontalis )

| The present study was undertaken to measure the biochemical profiles of freezable and nonfreezable ejaculates of Mithun. Fifty ejaculates (twenty five ejaculates each from freezable and nonfreezable ones) were collected from matured Mithun bulls. Biochemical profiles viz., alkaline phosphatase (ALP), acid phosphatase (ACP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic acid dehydrogenase (LDH), calcium (Ca), magnesium (Mg), zinc (Zn), chloride (Cl), citric acid (CA), total seminal plasma protein (TSPP), inorganic phosphorous (IP), fructose and cholesterol concentration in sperm were estimated. The result revealed that these parameters varied significantly (P< 0.05) between the freezable and non-freezable ejaculates. Freezable ejaculates has significantly (P< 0.05) higher ALP, ACP, LDH, Ca, Mg, Zn, citric acid, TSPP, IP, fructose and cholesterol concentration and significantly (P< 0.05) lower Cl concentration than the nonfreezable ejaculates. There was a correlation among the biochemicals such as ALP, ACP, LDH, Ca, Mg, Zn, CA, TSPP, IP, fructose, cholesterol and these biochemicals were negatively correlated with AST, ALT and Cl in both freezable and non-freezable ejaculates. It was concluded that most of the biochemical parameters were higher in freezable ejaculates than the non-freezable ejaculates, indicates that freezable ejaculates have has structural stability than the non-freezable ones that leads to freezable sperm has higher functional structures to move faster and in forward direction.


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insufficient number of breeding bulls, intensive inbreeding and lack of proper breeding management practices.Dwindling population of Mithun is a cause of concern which requires greater effort from all quarters to conserve the species bearing much effect on the socio-economic status of tribes.In Mithun natural breeding is being followed with accompanied limitations such as cost and disease transmission.Under such circumstances, wider application of artificial breeding of Mithun appears a viable option for conserving the species.
The seminal plasma is a highly complex biological fluid, secreted from various accessory sex glands and testes and contains various biochemical constituents such as cholesterol, sugars, proteins, metabolic, intra cellular and antioxidant enzymes, mineral elements (Kulkarni et al., 1996).The constituents of seminal plasma have various functions such acquisition of motility, capacitation, acrosome reaction and fertilizing capacity of spermatozoa (Kulkarni et al., 1996).Several studies were conducted on composition of seminal plasma in different domestic animal species (Mann, 1964;Dabas et al., 1984;Dhami and Sahni, 1994).Further, perusal of literatures revealed meagre information on the composition of seminal plasma in Mithun Bulls.Therefore, the study was designed to measure the biochemical composition of seminal plasma in Mithun to aid in for future semen preservation.

MATERIALS AND METHODS
Ten apparently healthy Mithun Bulls of 3 to 5yr of age were selected from the herd in Mithun farm, NRC on Mithun, Jharnapani, Nagaland.The average body weight was 501 kg (493 to 507 kg) with good body condition (score 5-6) maintained under uniform feeding (farm schedule) and managemental conditions.A total of 50 ejaculates were collected through rectal massage method.These ejaculates were splited into freezable and non-freezable ejaculates based on the post thaw motility (Rao and Rao, 1996;Prasad et al., 2000).Ejaculates having post thaw motility 40% and above were considered as freezable ejaculates whereas non-freezable ejaculates were considered with a post thaw motility less than 40%.
The biochemical profiles viz., ALP, ACP, AST, ALT, LDH activity, Ca, Mg, Zn, Na, K, inorganic phospho-rous, chloride, fructose, total seminal plasma protein and total cholesterol of the semen were estimated by commercial available kits (Cayman chemical company, USA).
The results were analysed statistically between the freezable and non-freezable ejaculates with the student 't' test using the SPSS (version 15.0; SPSS, Chicago, IL) and expressed as the mean ± S.E.M. Significant difference values of (p<0.05) were considered to be statistically significant after arcsine transformation of percentage data.Correlation among the biochemical parameters was estimated with Pearson's correlation coefficient.Differences at (p < 0.05) were considered to be statistically significant.

RESULTS AND DISCUSSION
In the present study, the result revealed that estimated different biochemical profiles varied significantly (P<0.05) between the freezable and non-freezable ejaculates (Table 1).Freezable ejaculates has significantly (P< 0.05) higher ALP, ACP, LDH, Ca, Mg, Zn, citric acid, TSPP, IP, fructose and cholesterol and significantly lower Cl content than non-freezable ejaculates.There was positive correlation among the biochemicals such as ALP, ACP, LDH, Ca, Mg, Zn, CA, TSPP, IP, fructose, cholesterol and these biochemicals were negatively correlated with AST, ALT and Cl in both freezable (Table 2) and non-freezable (Table 3) ejaculates.The present information may aid in getting good quality semen for artificial insemination by selecting freezable ejaculates based on biochemical constituents of seminal plasma.
Phosphatase enzymes such as ALP and ACP levels in seminal plasma are very important for sperm metabolism as well as sperm functions (Brooks, 1990).Therefore, estimates of these enzymes have been recommended as biomarkers for assessment of semen quality (Pesch et al., 2006).ALP in seminal plasma is primarily of testicular and epididymal origin and can be used as a clinical ejaculatory marker to differentiate azoospermia or oligospermia from ejaculatory failure (Turner and McDonnell, 2003).In the present study, the ALP was significantly decreased in the non-freezable ejaculates than the freezable ejaculates (Pesch et al., 2006).ACP is especially localized in corpus epididymidis, ductus epididymidis and vas deferens, but it is thought to be  an indicator for thesecretory function of prostate (Ciereszko et al., 1992).
Like phosphatse, AST and ALT are essential for metabolic processes, which provide energy for motility, viability and fertilizing ability of spermatozoa and these transaminase activities in the ejaculates are good indicators of semen quality as these enzymes measure stability of sperm membrane (Lopez et al., 1989).
Increasing abnormal sperm percentage in the ejaculate causes increased concentration of these transaminase enzymes in the extra cellular fluid which might be due to damage of sperm plasma membrane and leakage of enzymes from spermatozoa (Dogan et al., 2009).Moreover, increase in AST and ALT activities of seminal plasma of non-freezable ejaculates could be due to structural instability of the sperm or fragile nature of sperm membrane (Corteel, 1980) and less intactness of membrane of acrosome, plasma, mitochondria and flagella of the sperm.
LDH is an essential enzyme of almost universal distribution in the body which catalyses the reversible transamination of pyruvate to lactate.In semen, it is chiefly located in the mid-piece region (Dhami and Sahni, 1994).Like transaminases, LDH is also an es-sential enzyme responsible for metabolic processes, which provide energy for viability, motility, capacitation and fertilizing ability of spermatozoa (Sirat et al., 1996).It has been proposed that the seminal fluid LDH can be used as good indicator of sperm viability and membrane stability (Stamatiads et al., 1984).Pesch et al. (2006) reported that there was positive correlation among LDH activities, total sperm motility, progressive motility and liveability of sperm in fresh ejaculates, which may indicates that extracellular LDH ensures metabolism of spermatozoa.In the present study, extracellular LDH concentration was increased significantly (p< 0.05) in seminal plasma of freezable ejaculates (Table 1) than the non-freezable ejaculates.It has thus been proposed that higher LDH levels in seminal plasma of fresh semen can be used as a good indicator of higher motility, progressive motility and living sperm of ejaculates (Dube et al., 1982).The Ca, Mg, Zn and IP of ejaculate were significantly higher in freezable ejaculates than the non-freezable ejaculates, whereas chloride content was higher in non-freezable ejaculates in the present study (Table 1).Zn plays an essential role in normal testicular development, spermatogenesis and sperm motility (Wong et al., 2001).It is also a cofactor for a number of metalloenzymes in animals.Zn plays an important role in prostate, epididymal and testicular functions (Ebisch et al., 2003).Zn has been reported to influence the process of spermatogenesis (Wong et al., 2002), controls sperm motility (Wroblewski et al., 2003), stability of sperm membrane (Kendall et al., 2000), preserves the ability of sperm nuclear chromatin to undergo de-condensation and modulates sperm functions (Suruki et al., 1995).Hypozinkemia leads to gonad dysfunction, decreased testicular weight, atrophy of seminiferous tubules and complete cessation of spermatogenesis (Martin et al., 1994).
Calcium and Mg are required in many physiological processes as a regulator in all living cells, including spermatozoa.The presence of Mg 2+ and Ca 2+ ions is necessary for the last stage of capacitation following acrosome reaction and hyperactive motility of spermatozoa.Ca flux control through the spermatozoal membrane is essential for fertilization (Bailey and Buhr, 1993) and it was noticed that the inclusion of Ca 2+ with calcimine to isolated ram caudal spermatozoa caused stimulation of flagellar beat activity (Bradley and Forrester, 1982).But negative correlation between Ca concentration in seminal plasma and motility of bovine spermatozoa was also reported (Machal et al., 2002).In the present study, concentration of Ca and Mg were significantly higher in freezable than the non-freezable ejaculates.Calcium is positively correlated with sperm motility, viability and integrity of plasma and acrosomal membrane as Ca stabilizes the plasma membrane and influences its permeability and excitability.The higher correlation coefficient between seminal plasma Ca concentration and protein suggests that spermatozoon proteins as well as those of seminal plasma participate in extracellular Ca flow activation (Marques et al., 2000).Further, Ca is also required to stimulate the steriodogenesis in Leydig cells of the testis.Deficiency of Ca in the testes or accessory gland will leads to adverse effects on the normal function of the reproductive system and spermatogenesis (Kaplan et al., 1995).
Chloride levels were significantly (p< 0.05) higher in the non-freezable ejaculates than the freezable ejaculates (Table 1).The excess chloride could be toxic to bovine sperm which is neutralised by increased sodium levels (Dhami and Sahni, 1994).This could explain the reason for increased chloride in non-freezable ejaculates.In non-freezable ejaculates, seminal plasma fructose and metabolic enzyme concentra-tions are also low whereas chloride concentration is high (Hirsch et al., 1991).The low protein and high chloride content of the secretion is rather hostile to sperm.It is known that modulation of a variety of ion channels (like Cl) of spermatozoa is a characteristic event associated with capacitation and acrosome reaction of mammalian spermatozoa (Barrier-Battut et al., 2002).Hence, increasing the Cl level in seminal plasma may play a role in infertility.
TSPP in the seminal plasma fluid influence various functions of the sperm such as capacitation, acrosome reaction, motility, DNA integrity and fertilization of the oocyte (Moura et al., 2007).The level of TSPP in fresh semen of freezable ejaculates was significantly higher than the non-freezable ejaculates (Table 1).But some specific proteins might be responsible for the quality and freezability of semen which could not be differentiated between the two groups simply by estimating the total seminal plasma protein.Moreover, no report has so far been available on total seminal plasma protein level of freezable and non-freezable ejaculates in Mithun bull.Dhami and Sahni (1994) reported low mean protein value of static ejaculate than the motile ejaculate which supports the higher numerical value of TSPP in the highly motile samples in the present study.Similarly, Singh et al. (1989) reported a positive association of the protein values in the semen with its freezability.The reason could be the great importance of protein for motility and survival of spermatozoa during storage (Singh et al., 1989;Moura et al., 2007).Seminal plasm proteins are also known to have protective action towards sperm against lipid peroxidation (Schonek et al., 1996).But Mohanty (1999) reported higher TSPP in poor freezable bulls than the good freezable bulls.This variation could be due to different selection criteria of freezable and non-freezable ejaculates, frequency of collection, method employed for estimation, age and number of bulls studied and season (Dhami and Sahni, 1994).
Fructose and citric acid are reported to play important roles in sperm motility and concentration, particularly with regard to energy metabolism, through glucose utilization (Videla et al., 1981).Fructose is one of the major energy yielding nutritive substrates present in seminal fluid (Gonzales et al., 1997).It is secreted from the seminal vesicles and the accessory sex glands.Fructose is the major carbohydrate found in seminal plasma, provides over half the spermatozoa carbohy- drate consumption and appears essential for normal sperm motility.In the present study, the fructose and citric acid concentration were reduced significantly in non-freezable ejaculates (Table 1) as indicates that the functions of accessory sex glands was affected as seminal fructose is positively correlated with semen volume and sperm motility (Saeed et al., 1994).The determination of fructose itself is of particular significant because there is a direct relationship between the fructose level in seminal plasma and the testosterone function of the interstitial cells of Leydig.Fructose values which fall below normal may be a consequence of inflammatory condition in the prostrate or seminal vesicles or structural abnormality of the seminal vesicles and their ducts (Schirren, 1983).
It was concluded from the study that most of the biochemical parameters were significantly higher in freezable ejaculates in comparison to the non-freezable ejaculates of Mithun.This indicates that freezable sperm has structural stability than the non-freezable sperm that leads to freezable sperm has higher functional sperm structures to move faster and forward direction.

Table 1 :
Mean (±S.E.) biochemical attributes of freezable and non-freezable Mithun semen b Means with different superscript within rows differ significantly (P < 0.05); n= Number of ejaculates