This study has been conducted to evaluate the effect of inclusion of different levels of egg yolk (12%, 16% and 20%) in Tris-diluent on the viability, progressive motility, membrane integrity and acrosomal damage of Hariana bull spermatozoa at various stages of cryopreservation. 32 seminal ejaculates from 4 bulls (8x4) were subjected to cryopreservation and thawing process and evaluated with various tests at three different stages of freezing and thawing process (After dilution, after equilibration and after thawing). Statistical analysis was done as per the standard procedure (Snadocor and Cocharan, 1967). The mean percentage of live spermatozoa, progressive motile, HOST positive and sperms with intact acrosome were significantly higher (P<0.01) in diluents containing 16% and 20% egg yolk than in diluents having 12% egg yolk after dilution. While there was no significant difference in seminal attributes diluted with 16% and 20% egg yolk after dilution. Similar type of pattern was obtained after equilibration as well as after thawing. In conclusion, tris diluent containing 16% and 20% egg yolk provide better protection to sperms during cryopreservation than diluents containing 12% egg yolk. Furthermore concentration of egg yolk can be reduced from 20% to 16% in tris-diluent without any significant damage to sperms.
Cryopreservation, a non-physiological method, involves a high level of adaptation of biological cells to the osmotic and thermal shocks occurring during dilution, cooling, freezing and thawing process (Holt, 2000 b). More than 50% of the spermatozoa in the semen samples are usually injured by the cryopreservation process (Watson, 1995). When spermatozoa subjected to a rapid reduction in temperature from 25 to 5ºC (White, 1993), this produces cold shock, a membrane transition phase behavior exhibited by biological membranes (Watson and Morris, 1987). Further, it is one of the major causes of reduced motility after freeze-thawing (White, 1993), which often results in swelling and blebbing of the acrosomal membrane and disruption and/ or increased permeability of the plasma membrane (Ferna´ndez-Santos et al., 2006). In protecting the spermatozoa from such effects, the semen is generally being diluted in extender containing protective substances such as cryoprotectants (Situmorang, 2002) like egg yolk which can reduce the problem (White, 1993). Egg yolk is a routine component while diluting semen in the extenders in most of the cryopreservation protocols in many species including bovine (Shannon and Curson, 1983; Priyadharsini et al., 2011). The active constituent/component of egg yolk is low-density lipoprotein (LDL) fraction that are responsible for protecting sperm cells from cold shock (Watson and Martin, 1975) and also the cell membrane during freezing and thawing (Aboagla and Terada, 2004).
Despite having the beneficial effects, different concentrations of egg yolk in the extender can influence the parameters of semen viscosity and sperm motility (Hirai et al., 1997). However, recent reports suggest that an increase in egg yolk concentration in the TEST diluents decreases Sperm Motility Index (Garde et al., 2008) and negatively affects preservation of motility and acrosome integrity in Tes-Tris based diluent (Holt et al., 1996) during cryopreservation of semen in Mohor and Cuvier’s Gazelle species, respectively. Further, it is also reported that higher egg yolk concentrations decreases post thaw motility of buffalo semen when frozen in Tris based dilutor (Kumar et al., 1994). Hence, there is a need to work out the optimal level of egg yolk in the cryopreservation protocol for the beneficial effect as it may vary from species to species (Situmorang, 2002) and also from laboratory to laboratory. In the present study, we investigated the effect of three levels of egg yolk in Tris-dilitor on viability, motility, membrane integrity as well as acrosomal damage during cryopreservation and after thawing in Haryana bull semen.
Material and Methods
Four Hariana cattle bulls (aged 4.5 to 5.5 years, 450 to 500 kg body weight) managed under proper farm condition were used as semen donors. The eight ejaculates from each of 4 bulls with single ejaculate collected biweekly was screened and those having more than 70 per cent progressive motility, 80 per cent live spermatozoa and 600 millions/ ml sperm concentration were used for cryopreservation. Eight ejaculates from each bull were processed for cryopreservation. Semen was diluted in Tris buffer extender (Tris buffer: 3.028 gm, Citric acid monohydrate: 1.675 gm, Fructose anhydrous: 1.250 gm, Penicillin G. sodium: 1 lac unit, dihydrostreptomycin sulfate: 100mg and double distilled water up to 100 ml) containing 7% glycerol with three different concentrations of egg yolk i.e. 12%, 16% and 20%. Dilution was made in two steps i.e. 50% dilution without glycerol at room temperature and 50% dilution with glycerol at 40 C followed by filling and sealing of straws using automatic filling and sealing machine (MRS-1 Dual, IMV France). The semen filled straws were equilibrated at 40C for 5 hr. Equilibrated semen straws were placed in programmable biological freezer (Win 3t Digit cool # 11011701, IMV France) for LN2 vapor freezing at a freezing rate of 5ºC/min (4 to -10ºC), 40ºC/min (-10 to -100ºC) and 20ºC/min (- 100 to -140ºC) with entire freezing time of 7 minutes (40C to -1400C). The cryopreserved straws were stored into LN2. After 24 hours, the frozen 32 straws (8 x 4 bulls= 32) were thawed in thawing unit (IMV, France) maintained at 370C with holding time 45 seconds. The semen samples were subjected to various tests i.e. per cent live spermatozoa, progressive motility, Hypo Osmotic Swelling (HOS) test and acrosomal integrity at three various stages of freezing and thawing process (After dilution, after equilibration and after thawing). Statistical analysis was done as per the standard procedure (Snadocor and Cocharan, 1967). The means were compared using analysis of variance (ANOVA, one way and Duncan’s Multiple Range Test (DMRT).
Assessment of Semen
Viability of the spermatozoa was assessed using a phase contrast microscope (x1000) under oil immersion by examining Eosin-Nigrosin stained semen smear. Eosin penetrated spermatozoa (pink color) either partially or fully was considered as dead spermatozoa. An overall 200 spermatozoa were counted from different fields and viability percentage was calculated by dividing the number of viable spermatozoa with total number of evaluated spermatozoa.
The progressive motility was assessed subjectively using a phase contrast microscope (x400) by examining the thin film of the semen placed on a warm glass slide and thermostatically controlled warm stage of the microscope.
Spermatozoa Membrane Integrity Test
The test was done as described by the method of Correa and Zavos (1994). The semen was submitted to HOS test. Briefly, a volume of 10 µl of semen was added to 990 µl of hypo-osmotic solution (150 m Os ml/L) composed from fructose and sodium citrate and incubated at 370C for 60 minutes. Immediate after incubation, one drop (10 µl) of semen was placed on a glass slide, covered with cover slip and evaluated under phase contrast microscope (x400). At least 200 spermatozoa were counted from randomly selected microscopic fields. The spermatozoa showing swollen or coiled tail were designated as HOS test positive spermatozoa and recorded. The percentage of HOS test positive spermatozoa were calculated against Control having 10 µl of semen added in 990 µl of normal saline.
Intact acrosome was assessed using a phase contrast microscope (x1000) under oil immersion by examining Giemsa stained semen smear. The 10% buffered formal saline fixed semen smear (for 10 minutes at 370C) was stained with buffered working giemsa solution (Giemsa stock- (Giemsa stain : 3.8 gm, absolute alcohol: 375 ml, glycerol: 125 ml) , Giemsa working ( 3 ml Giemsa stock + 2 ml Sorenson’s phosphate buffer (pH 7) + 35 ml distilled water) ) for 90 min at 370C in an incubator. The smear was then examined for acrosome intactness as per the method of Watson (1975).
Result pertaining to the viability of the spermatozoa, progressive motility, membrane integrity and acrosomal integrity of the spermatozoa are presented in the Fig. 1, 2, 3, and 4 respectively. Perusal of the data of spermatozoa viability revealed that the percent live spermatozoa are significantly higher (P<0.01) in the semen sample diluted in 16% and 20% egg yolk than diluted in 12 % egg yolk after dilution, after equilibration and post thaw. However no significant difference was observed between 16% and 20% throughout the process of freezing i.e. after dilution, after equilibration and post thaw (Fig. 1).
Result, pertaining to the progressive motility of the spermatozoa, exhibited that immediate after dilution, the percent progressive motile spermatozoa are significantly higher in the semen diluted in 16% and 20% egg yolk than diluted in 12 % egg yolk whereas no significant difference was observed between semen samples diluted in 16% and 20%. Same pattern was also seen after equilibration as well as after thawing (Fig. 2). Observations from the obtained data regarding membrane integrity of the spermatozoa, indicated that immediate after dilution, the percent HOS positive spermatozoa are significantly higher in the semen diluted in 16% and 20% egg yolk than diluted in 12 % egg yolk.
However no significant difference was observed between semen samples diluted in 16% and 20% of egg yolk. Same pattern was also seen after equilibration as well as after thawing (Fig. 3).
Acquired data exhibited that immediate after dilution, the percent spermatozoa with intact acrosome are significantly higher in the semen diluted in 16% and 20% egg yolk than diluted in 12 % egg yolk after dilution, after equilibration and post thaw whereas no significant difference was observed between semen samples diluted in 16% and 20% egg yolk. Similar type of pattern was seen throughout the process of freezing i.e. after dilution, after equilibration and post thaw (Fig. 4).
Egg yolk is a common content of semen freezing extenders for most of the domestic animals including buﬀalo (Sansone et al., 2000) and many exotics species (Holt, 2000). It is accepted that the sperm protection during cryopreservation is determined by the phospholipids (lecithin) and low density lipoproteins (LDL) (Medeiros et al., 2002; Purdy, 2006) contained in egg yolk. Egg yolk acts on the cell membrane, having a greater effect in bull than ram spermatozoa (Barbas and Mascarenhas, 2009). It is suggested that sperm membrane is adhered with LDL which provides protection to sperm by stabilizing the membrane (Andrabi, 2009) as well phospholipids present in LDL protect sperm by forming a protective ﬁlm on the sperm surface or by replacing sperm membrane phospholipids that are lost or damaged during the cryopreservation process (Graham and Foote, 1987). LDL may also resists the harmful proteins present in seminal plasma thus improves the freezing ability of sperm (Bergeron and Manjunath, 2006). The exact mechanism by which egg yolk protects the spermatozoa during cryopreservation process is unknown (Bathgate et al., 2006).
Review of literature reveals that generally egg yolk is used at a concentration of 20% in semen extender (Sansone et al., 2000; Andrabi et al., 2008). No doubt about the protective action of egg yolk but some studies have shown the deleterious effect when it was used in higher concentration along with toxicity (amino acid oxidase activity) of dead spermatozoa resulting in lower post-thaw spermatozoal quality (Shannon, 1972). High proportions of egg yolk depress sperm motility and increase the incidence of acrosomal damage in several species, such as brindled gnu (Connochaetes taurinus) (Watson, 1976), water buffalo (Bubalus bubalis) (Kumar et al., 1993) or ram (O. aries) (Watson and Martin, 1975). Several substances in EY have been proved to inhibit cells respiration and therefore lead to a decrease in number of motile cells in bull (Moussa et al., 2002). Recent studies have demonstrated that some egg yolk components, not contained in the low density lipoprotein (LDL) fraction, are toxic for cells during the dilution, prior to freezing and might exert a negative effect on the fertilizing ability of epididymal spermatozoa of bull (Amirat et al., 2005). In this regard, Sahni and Mohan (1990) studied different levels of egg yolk in extender for cryopreservation of buffalo semen and 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. Shannon and Curson (1983) also reported that egg yolk levels in diluents can be reduced from 20 to 2% without significant reduction in conception rate of highly-diluted bovine semen. Similarly Kumar et al. (1994) found that 5% egg yolk prove better than 10% and 20% while Singh et al. (1999) concluded 10% concentration of egg yolk was superior for freezing of buffalo semen with regards to pre-freeze and post-thaw sperm motility and survivability of spermatozoa. Detrimental effect of high concentration of egg yolk on sperm quality has also been reported in Spanish ibex (Julia´n Santiago-Moreno et al., 2006), Iberian Red deer (Ferna´ndez-Santos et al., 2006) and Cuvier’s gazelle (Grade et al., 2008). M .Valenica et al. (1977) put the view that lower levels of egg yolk may be necessary for greatest post-thaw motility when spermatozoa are frozen in continental or French straws and concluded that post thaw motility is better at 16 % than 32% as well as 8%. In one of the study by Van Denmark et al. (1957), it was found that percentage of motile spermatozoa of bull after freezing and thawing was similar for 15% and 30%, but was significantly lower at egg yolk concentrations above and below this range. Swanson (1949) reported the use of 20% egg yolk concentration that has a good effect on bovine sperm motility and livability. In a study quail and chicken egg yolk are compared in concentrations of 0, 2, 5, 10, 15 and 20% and they found that 10% quail egg yolk concentration improved the percentages of motile and progressively undulating spermatozoa in comparison to the 10% chicken egg extender (Trimeche et al., 1997). Similarly, Rahman et al. (2012) concluded that replacing chicken egg yolk extender with pigeon egg yolk extender in same concentration (20%) didn’t improve post thaw semen quality. However, reducing the concentration of pigeon egg yolk extender from 20% to 5% had adverse effects on post-thaw quality of Sahiwal bull semen.
There are so many literatures available regarding the effect of egg yolk on semen cryopreservation and most of them indicated that increased level of egg yolk more than 20% negatively affect the frozen-thawed semen quality. In the present study our results suggest that the percentage of egg yolk can be reduced to 16% without affecting much in the seminal characters of Hariana bull as viability, progressive motility, membrane and acrosomal integrity of sperms. This, on the other hand will also help in restricting contaminants (checking microbial growth) as well ease microscopic evaluation of sperm quality.