Printer Friendly

Seminal plasma heparin binding proteins improve semen quality by reducing oxidative stress during cryopreservation of cattle bull semen.

INTRODUCTION

Sperm cryopreservation is an important tool for assisted reproductive techniques and is the most efficient way for storing germ plasm (Olaciregui et al., 2014). Cryopreservation has been applied to various species, including humans, swine, cattle, cats, and dogs (Mota Filho et al., 2014). Although sperm cryopreservation preserves sperm motility, metabolic functions, and fertility, but, still the freeze-thawing process causes irreversible damage to spermatozoa and reduces fertility (Ardon and Saurez, 2013). Even though cryopreservation helps in prolonged storage of semen, but cryo-capacitation is one of the major factors associated with reduced longevity and poor survivability of cryopreserved spermatozoa in female reproductive tract (Watson, 2000), resulting in reduced fertility of frozen-thawed semen. Even with optimized protocols, 40% to 50% of the sperms do not survive after cryopreservation due to irreversible damage (Watson, 2000). Several studies have focused on identifying damages during freezing and thawing, tests to screen sperm quality of frozen-thawed semen, evaluation of alternative cryoprotectants and other additives, and freezing procedures to improve sperm viability and fertility. Recently, Yoon et al. (2015) proposed that freezing/thawing steps are the most critical in cryopreservation and may provide a logical ground to understand the process of cryo-damage.

Currently, many diverse proteins are being tested that may increase the fertilization capability of sperm and heparin binding proteins (HBPs) are among these proteins. HBPs have been viewed as favouring capacitation, acrosome reaction, and altering the immune system response toward the sperm. The HBPs are produced by the accessory glands of the male under the control of androgens (Miller et al., 1990) and secreted within the seminal fluid (Nass et al., 1990). The HBPs bind to the epididymal sperm and increase the ability of the acrosome reaction in response to the heparin and other proteins of the zona pellucida (Ax et al., 2002). Absorption of isolated proteins from the seminal plasma can reduce the damage that cold shock can have on the sperm membrane (Barrios et al., 2000). Mogielnicka-Brzozowska and Kordan (2011) mentioned the possibility of adding specific plasma proteins to sperm for the retention of features responsible for the efficient fertilization after storage. HBPs protect sperm from stress of freezing and thawing and maintained intracellular protein homeostasis (Shi et al., 1998). HBPs also protect sperm from lipid peroxidation during cryopreservation (Kumar et al., 2008). Preliminary studies were done by Alvarez-Gallardo et al. (2013) with the recombinant proteins indicated that addition of rFAA and rTIMP-2 to semen before cryopreservation help stabilize the acrosome membrane of the bovine sperm by decreasing post-thaw acrosome damage. Hence the present study was conducted with objective to assess the effect of HBP on sperm function tests at pre-freeze and frozen-thawed phases of cryopreservation.

MATERIALS AND METHODS

Procurement of semen sample

Three ejaculates from each of six healthy cross cattle bulls (Holstein-Fresian x Red Dane x Sahiwal) were procured from GADVASU, Dairy Farm and Semen bank, Bhattian, Khanna, Ludhiana, India. The bulls were kept under loose housing system (covered area, 3.6 m x 3.0 m and uncovered area, 7.5 m x 3.0 m) and standard feeding schedule along with ad libitum green fodder and water availability and standard management conditions. Semen was collected twice a week through artificial vagina method. None of the bulls selected for this study had any preceding physical abnormalities.

A good quality fresh ejaculates (1 to 2 mL) with minimum initial motility of [greater than or equal to]60%, mass activity of 3+ to 4+, abnormal morphology of [less than or equal to]25% and sperm concentration of >750 x [10.sup.6] sperms/mL of each bull were processed for cryopreservation. Seminal plasma was separated by centrifuging pooled semen of six bulls at 3,000 rpm for 5 minutes and was stored at -20[degrees]C till use.

Purification of HBPs by affinity chromatography

Sepharose (Sigma-Aldrich) was activated and heparin was bound as per the protocol of Farooqui (1980). The chromatography glass column (28 mm x 70 mm) was packed with heparin-sepharose media up to the mark at the top of the column and allowed to settle for 3 to 4 h. Thereafter, the column was equilibrated for 1 h with 10 mM Tris HCl (pH 7.4). About 1.0 mL of pooled seminal plasma (filtered through 0.22 p filter) was loaded and circulated through the column for 15 minutes for absorption of HBPs to the heparin bound resins. The non-HBPs were washed out with 10 mM Tris HCl and the HBPs were eluted with 1 M NaCl at a flow rate of 1 mL/min in the tubes racked in a fraction collector. The recovered HBPs fractions were pooled in agreement with the observed curve, obtained from optical density (280 nm), detected by UV monitor, attached to the fraction collector (Manaskova et al., 2002). The pooled fractions of HBPs were concentrated by spinning through protein concentrators (Millipore, Billerica, MA, USA, 3 kDa) and analyzed for protein content (Lowry et al., 1951).

Conjugation of fluorescein isothiocyanate fluorochrome with purified HBP and confirmation of incorporation of SP-HBP to semen

Conjugation of fluorescein isothiocyanate (FITC) fluorochrome to SP-HBPs was done by using a kit (Life technology, Carisbad, CA, USA) as per the protocol provided with the kit. About 20 pg of purified HBPs was added to 1.0 mL of neat semen and incubated at 37[degrees]C for 20 minutes. Semen was washed thrice with phosphate buffered saline (PBS), pH 7.4 by centrifugation at 3,000 rpm for 5 min. Pellet was suspended in 1.0 mL of PBS and a drop of semen on a glass slide covered with coverslip was observed at 400 x under fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) using blue filter at 480 nm.

Dilution and cryopreservation of semen

Three doses of SP-HBPs i.e. 10, 20, 40 pg/mL semen were standardized to find out the optimum dose and 20 pg/mL was found to be an optimum dose. Semen was divided into two fractions: one fraction, supplemented with SP-HBP and other as such (control), were incubated at 37[degrees]C for 20 min. After incubation, both fractions were diluted with sodium citrate extender. The pre-freezing sample was kept at 4[degrees]C in cold handling cabinet for equilibration and aliquot for freezing was filled and sealed by manual filling and sealing method. The filled and sealed straws were cryopreserved in liquid nitrogen by following standard procedure. Semen at pre-freeze and frozen-thaw phase was analyzed for sperm function (motility, viability and Hypo osmotic swelling test [HOST]), acrosome damage, acrosome reaction and lipid peroxidation [LPO]) as detailed below.

Individual motility

About 10 [micro]L of semen was placed on a pre-warmed (37[degrees]C) glass slide, covered with a cover slip and placed on the pre-warmed stage (Magnus Mumbai, Maharashtra, India) attached to a phase contract microscope (Olympus CX21FS1, Japan). Linear progressive motility was assessed by subjective method viewing 10 fields with the aid of closed-circuit television attached to the microscope (400 x). A total of 200 spermatozoa were observed under phase contrast microscope (Olympus, Japan) at 400* in different fields and percentage of motile spermatozoa was calculated.

Sperm viability

Sperm viability was assessed by Syber green/propium iodide (Life technologies staining kit, USA). A total of 200 live (green) and dead (red) spermatozoa were counted in different fields and percentage of viability was calculated (Figure 1).

Hypo-osmotic swelling test

Briefly, 10 [micro]L of semen was incubated in 100 [micro]L of hypo-osmotic solution at 37[degrees]C for 30 min. A total of 150 spermatozoa with coiled and un-coiled tails were counted in different fields (Jeyendran et al., 1984). A control was also run in PBS (pH 7.4). The number of spermatozoa with coiled tailed/swollen heads in PBS was deducted from the number in hypo-osmotic solution and the resultant figure was taken as the Hypo-osmotic swelling (HOS)-reactive spermatozoa.

Acrosomal damage

Acrosome damage was assessed by chlortetracycline cysteine stain (CTC, Figure 2) (Ward and Storey, 1984). Briefly 25 [micro]L of sperm suspension was mixed with 25 [micro]L of 750 mM CTC in 20 mM Tris-HCL, 130 mM NaCl and 5 mM cystein, incubated at 37[degrees]C for 30 min. After adding 100 [micro]L of 12.5% paraformaldehyde and one drop of 0.22 M 1, 4-Diazabicyclo[2.2.2]octane, a drop of sperm suspension was observed under fluorescence microscope (Olympus, Japan) at 400 x using blue filter at 480 nm. At least 200 spermatozoa with normal (whole sperm head with bright fluorescence) and cryo capacitated sperm (acrosome-intact sperm with fluorescence ring at equatorial segment) and acrosome-reacted (sperm with fluorescence on postacrosomal region) were counted in different fields at 400 x and percentage of cryo-capacitated and acrosome-reacted spermatozoa was calculated (Figure 2).

In vitro capacitation/acrosome reaction

Frozen-thawed spermatozoa washed twice with tyrode albumin lactate pyruvate medium (TALP, 92.9 mM NaCl, 4 mM KCl, 25.9 mM NaHCOs, [Na.sub.2]HP[O.sub.4], 10 mM Ca[Cl.sub.2] x 2[H.sub.2]O, 0.5 mM Mg[Cl.sub.2] x 6[H.sub.2]O, 1.3 mM sodium pyruvate, 7.6 mM sodium lactate and 20 mM 4-2-hydroxyethyl-1-piperazine-ethane-sulfonic acid, pH 7.4 were subjected to swim up and only motile spermatozoa were incubated in TALP supplemented with 0.6% bovine serum albumin, 5 mM glucose and 165 units of heparin for 4 hrs at 37[degrees]C in an incubator (Fraser et al., 1995). Capacitation/acrosome reaction was assessed by counting 200 spermatozoa in CTC stained slides under fluorescence microscope at 400 x (Olympus, Japan).

Lipid peroxidation

Lipid peroxidation was estimated by the end point generation of malondialdehyde (MDA) determined by the thiobarbituric acid (TBA) test (Buege and Steven, 1978). Briefly, extended spermatozoa ([40 to 70] x [10.sup.6] cells) in 0.5 mL of cold 10% (wt/vol) chilled trichloroacetic acid to proliferate proteins. The precipitate was pelleted by centrifugation (6,000 rpm for 10 min) and 1 mL of 0.375% (wt/vol) TBA was added to the supernatant and kept in a boiling water bath at 100[degrees]C for 10 minutes. After cooling, the absorbance was read at 532 nm (UV-VIS spectrophotometer, systronics, Ahmedabad, Gujrat, India).

MDA production was calculated by the formula:

[OD at 532 nm x volume of assay mixture]/[molar extinction coefficient x volume of sample]

* Molar extinction coefficient = (1.56 x [10.sup.5] M-1C-1) Statistical analysis

The data is presented as mean [+ or -] standard error of the mean and analyses were performed with CPCS1 program package. One way analysis of variance was applied to evaluate the level of significance at 5% between treatments.

RESULTS AND DISCUSSION

Purification of HBPs from seminal plasma and verification of binding of SP-HBP to spermatozoa

Graphic image of HBP purified from pooled seminal plasma of cross-bred bulls is shown in Figure 3, which indicated two peaks of unbound (non-HBP) and bound proteins (HBP). Addition of 20 [micro]g/mL of SP-HBP to neat semen and incubation at 37[degrees]C for 20 min before cryopreservation resulted in its binding mainly to head and mid piece. But binding was more on post-acrosomal cap in majority of the spermatozoa (Figure 4). A study done by Cheema et al. (2015) on natural localization of HBPs in cross-bred bull spermatozoa using anti-AZU-1 revealed that these proteins are mainly localized on acrosomal cap.

Effect of SP-HBP on sperm function tests

Standardization of dose of SP-HBP : Effects of different doses of SP-HBP on percent motility, viability, HOST, acrosome damage, in vitro capacitation and LPO in pre-freeze and frozen-thaw semen are given in Table 1. Individual motility (%), viability (%), HOST (%) were found to be higher in pre-freeze and post-thaw semen supplemented with 20 gg/mL of SP-HBP as compared to 0, 10, and 40 gg of SP-HBP. Percentage of in vitro capacitated/acrosome reacted spermatozoa was higher only in frozen-thawed semen supplemented with 20 gg/mL SPHBP as compared to 0, 10, and 40 gg. Acrosome damage (%) and MDA (gM/109 spermatozoa) showed reduction in semen supplemented with 20 gg/mL of SP-HBP in comparison to 0, 10, and 40 gg SP-HBP at both phases of cryopreservation. Increase in motility, viability, in vitro acrosome reaction and decrease in LPO (MDA) in semen supplemented with SP-HBP was statistical significant (p<0.05). Manjunath et al. (2002) also suggested that at a greater concentration of bovine seminal plasma proteins, more cholesterol and phospholipids are removed from the sperm membrane, resulting in its destabilization that decreases sperm resistance to cold shock and freezing.

Harshan et al. (2006) also postulated that higher loss of semen parameters with higher amount of HBP could be due to an increased cholesterol efflux in the spermatozoa which led to more pronounced cryo-damage. Therefore, effects of 20 [micro]g/mL of SP-HBP on semen cryopreservation of six bulls were evaluated.

Effect of 20 [micro]g SP-HBP on sperm function and fertility tests of six bulls

Motility, Viability and HOST: Mean percentage of motile, live and HOS responsive spermatozoa showed a variation among six cross breed bulls, which ranged from 61.7% [+ or -] 1.7% to 70 [+ or -] 2.9%; 55.7% [+ or -] 2.8% to 73.0% [+ or -] 6.6%; 16.0% [+ or -] 0.7% to 36.1% [+ or -] 8.0%, and 41.7% [+ or -] 4.4% to 58.3% [+ or -] 6.7%; 38.2% [+ or -] 3.1% to 59.5% [+ or -] 2.2%; 8.8% [+ or -] 4.6% to 27.5% [+ or -] 7.5% at the time of pre-freezing and freezing-thawing, respectively (Table 2). Decrease observed in percent motility, viability and HOST from pre-freeze to post thaw phase of cryopreservation was significant (p<0.05). Lessard et al. (2000) were of the opinion that sperm viability decrease by 50%, whereas, fertilizing capacity is affected by a factor of sevenfold after cryopreservation. However, incorporation of SP-HBP to the sperm surface resulted in an increase of 5.0% to 10.0%; 3.6% to 7.8%; 4.8% to 22.1%, and 1.6% to 8.3%; 4.2% to 11.0%; 3.0% to 12.0% in motile, live and HOS positive spermatozoa at pre-freeze and frozen-thaw phase of cryo-preservation as compared to control, respectively. However, increase in only motility and HOST due to supplementation of SP-HBP was significant (p<0.05) at both phases of cryopreservation. Harshan et al. (2006) revealed that 40 pg dose of HBP improved the motility, HOST and cervical mucus penetration test at pre-freeze phase of buffalo semen cryopreservation, but they did not observe positive effect of this dose on frozen-thawed semen. Barrios et al. (2000) also demonstrated that the absorption of isolated proteins from seminal plasma may reduce the cold shock effect.

Acrosome damage: Two stages of acrosome damage were revealed by CTC staining i.e. partial (cryo-capacitated) and complete (cryo-acrosome reacted). These two phases of sperm due to cryo-injury are demonstrated as premature capacitation and spontaneous acrosome reaction (Srivastava et al., 2013). Mean percentage of cryo-capacitated and acrosome reacted spermatozoa showed a variation among the bulls and ranged from 20.1% [+ or -] 1.1% to 30.6% [+ or -] 1.5% and 5.3% [+ or -] 0.3% to 14.3% [+ or -] 4.1% at pre-freeze phase, which was further significantly (p<0.05) increased to 25.6% [+ or -] 4.5% to 37.5% [+ or -] 0.7% and 10.9% [+ or -] 1.3% to 21.1% [+ or -] 0.5% in frozen-thaw phase of cryopreservation in six bulls, respectively (Table 3). Altered mitochondrial function, reduction in motility and failure of chromatin condensation, all of which influence the viability and fertility of sperm have also been reported due to induction of pre-mature acrosome reaction (Watson, 2000; Chaveiro et al., 2006). Acrosomal cap is of utmost importance for fertilization and is related to fertility. Spermatozoa with higher motility cannot be fertile due to acrosomal loss (Raval and Dhami, 2010).

Acrosome is required to undergo biochemical changes in the female reproductive tract before fertilization and premature loss of acrosome and hence loss of enzymes will affect the fertilization process. Supplementation of SP-HBP to cattle bull semen resulted in a non-significant (p<0.05) reduction of 1.0% to 1.4% and 0.6% to 17.9% in acrosome damage at pre-freeze and frozen-thaw phase, respectively. Our study revealed a reduction in cold shock effect on treating the spermatozoa with SP-HBP. Result of a study by Mogielnicka-Brzozowska et al. (2011) also indicated that zona binding proteins of boar seminal plasma have a shielding effect on the plasma membrane and the acrosome of spermatozoa by protecting their structures against the damage caused by cold shock. Mogielnicka-Brzozowska and Kordan (2011) mentioned that addition of specific plasma protein to sperm probably may retain the features responsible for the efficient fertilization after storage.

In vitro capacitation and acrosome reaction: A variation in response of frozen-thawed spermatozoa to in vitro capacitation and acrosome reaction was also observed irrespective of treatment among the six bulls. Incubation of frozen thawed spermatozoa of six bulls in TALP energy medium resulted in 36.6% [+ or -] 2.1% to 45.6% [+ or -] 1.8% capacitated and 23.9% [+ or -] 2.2% to 31.7% [+ or -] 1.7% acrosome reacted spermatozoa (Table 4). Incorporation of SP-HBP enhanced the percentage of capacitated (41.9% [+ or -] 1.7% to 48.5% [+ or -] 0.2%) and acrosome reacted (26.6% [+ or -] 2.5% to 33.5% [+ or -] 5.4%) spermatozoa in frozen-thawed semen of six bulls. It indicated a significant (p<0.05) increase of 6.2% to 11.4% in capacitated/acrosome reacted spermatozoa upon binding of SP-HBP to the sperm membrane. Arangasamy (2010) performed in vitro capacitation of stallion spermatozoa in the presence of SP-HBP/heparin and found significant difference in treated and control groups. Heparin and heparin like molecules are known to effectively capacitate bovine sperm. High density lipoproteins (HDL) have also been shown to capacitate bull sperm (Therein et al., 1998) and HDL by binding to the bovine seminal plasma proteins (Manjunath et al., 1988). Addition of purified fraction of 15 to 17 kDa, 24 kDa, and 31 kDa HBPs (Miller et al., 1990) to epididymal sperm revealed the effect of heparin. They further conveyed that fraction containing 24 and 31 kDa proteins possessed greatest activity/unit of protein to induce acrosome exocytosis.

Lipid peroxidation: An increase of 35.8 to 108.1 [micro]moles of MDA/[10.sup.9] spermatozoa was noticed from pre-freeze to freezing-thawing process of cryopreservation (Table 5). MDA has been used in biochemical assays to monitor the degree of peroxidative damage in spermatozoa and exhibited an excellent correlation with the degree to which sperm function is impaired in terms of motility and the capacity for sperm-oocyte fusion (Sidhu et al., 1998). Increase in MDA production due to freezing-thawing was non- significantly (p>0.05) less (23.1 to 86.9 pmoles/109 spermatozoa) in semen supplemented with SP-HBP in comparison to control.

There was a non significant (p>0.05) decline of 5.8 to 40.1 pmoles/109 and 16.3 to 64.7 pmoles/109 spermatozoa in pre-frozen and frozen-thawed semen supplemented with SP-HBP, respectively. Karunakaran et al. (2012) added 25 pg of 28 to 30 kDa HBP to the frozen-thawed bull semen in control and H2O2 treated group and concluded that excessive H2O2 causes significant increase in LPO levels and 28 to 30 kDa HBP of sperm membrane helped in controlling the oxidative stress of sperm cells. Kumar et al. (2008) were also of the opinion that HBPs protected the buffalo bull spermatozoa from lipid peroxidation stress during cryopreservation.

Seminal oxidative stress develops as a result of an imbalance between reactive oxygen species and scavenging activities. Spermatozoa are particularly susceptible to the oxidative stress induced damage as their plasma membrane contains large quantity of polyunsaturated fatty acids. Cytoplasm of spermatozoa has low concentration of scavenging enzymes (Sharma and Agarwal, 1996). Moreover mature sperm is devoid of cytoplasm and intracellular antioxidant enzymes cannot protect the plasma membrane that surrounds acrosome and tail. Therefore, for their protection spermatozoa depend upon seminal plasma. Small amounts of reactive oxygen species (ROS) have been presented to be required for several functions of spermatozoa, but their excessive levels can negatively impact the quality of spermatozoa and impair the fertilizing capacity (Tvrda et al., 2011). Reactive oxygen species are usually formed during normal enzymatic reaction of inter and intracellular signalling (Agarwal and Allamaneni, 2006). Therefore, declined level of MDA production in the spermatozoa treated with SP-HBP may be due to its interference with enzymatic reaction of inter and intracellular signalling responsible for ROS production.

Oxidative stress affects the fluidity of sperm plasma membrane as well as integrity of DNA in the nucleus (Aitken, 1999). Increase in membrane fluidity leads to sperm capacitation. Therefore, higher level of acrosome damage (cryocapacitation like changes ) observed in frozen-thawed spermatozoa as compared to pre-frozen spermatozoa may be due to increase in ROS level during cryopreservation of semen, which was also evidenced from increased level of MDA production. Spermatozoa itself produce small amounts of ROS that are essential to many of physiological process i.e. capacitation, hyperactivation and sperm oocyte fusion (Aitken et al., 2003). Low level of ROS has also been shown to be essential for fertilization, acrosome reaction and motility. Production of higher level of ROS as observed by a rising level of MDA production in frozen-thawed spermatozoa was repressed by addition of SP-HBP. Therefore reduced level of oxidative stress in frozen thawed spermatozoa treated with SP-HBP may be responsible for higher percentage of motile, viable, HOS positive, in vitro capacitated/acrosome reacted spermatozoa and spermatozoa with intact acrosomes in pre-frozen and frozen-thawed semen of six cross bred bulls.

It was observed during the present study that cryopreservation of semen of six bulls resulted in reduced motility, viability, HOST and enhanced acrosome damage/oxidative stress with a variation among the bulls. Such a variation may be attributed to varying concentration of membrane cholesterol. Fresh spermatozoa with higher cholesterol content are expected to resist destabilization of membrane following cryopreservation (Srivastava et al., 2013). Since membrane integrity is altered with cryopreservation, spermatozoa with more cholesterol content prior to cryopreservation will also resist membrane damage and effect of membrane damage and cryocapacitation will be less evident in these bulls (Srivastava et al., 2013). Treatment of semen with SP-HBP improved motility, viability, HOST, acrosome integrity, in vitro capacitation/acrosome reaction by diminishing oxidative stress, but with a variation among the bulls. HBPs are acquired by spermatozoa at the time of ejaculation and have been associated with removal of cholesterol from sperm membrane during capacitation (Therien et al., 2001). Therefore, variation in response of spermatozoa to SP-HBP treatments may also be associated to varying concentration of sperm membrane cholesterol.

Our observations revealed that absorption of SP-HBP to the sperm surface reduced the damage that cold shock can have on the sperm membrane during the process of freezing, consequently as pointed out by Lessard et al. (2000), increase in fertilizing capacity of SP-HBP treated cryopreserved semen should be expected.

http://dx.doi.org/ 10.5713/ajas.15.0586

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

REFERENCES

Agarwal, A. and S. Allamaneni. 2006. Oxidative stress and human reproduction. In: Oxidative Stress, Disease and Cancer (Ed. K. K. Singh). Imperial College Press, New York, NY, USA. pp. 687-703.

Aitken, R. J. 1999. The Amoroso lecture the human spermatozoon--a cell in crisis? J. Reprod. Fertil. 115:1-7.

Aitken, R. J., M. A. Baker, and D. Sawyer. 2003. Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reprod. Biomed. Online 7:65-70.

Alvarez-Gallardo, H., M. E. Kjelland, J. F. Moreno, T. H. Welsh, Jr. and R. D. Randel. 2013. Gamete therapeutics: Recombinant protein adsorption by sperm for increasing fertility via artificial insemination. Plos One. 8:e65083.

Arangasamy, A. 2010. Effect of stallion seminal plasma proteins on in-vitro capacitation of equine spermatozoa. Ind. J. Anim. Sci. 80:394-397.

Ardon, F. and S. S. Sauarez. 2013. Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5. Reproduction 146:111-117.

Ax, R. L., H. E. Hawkins, S. K. DeNise, T. R. Holm, and H. M. Zhang. 2002. New developments in managing the bull. In: Factors Affecting Calf Crop: Biotechnology of Reproduction (Eds. M. J. Fields, R. S. Sand, and J. V. Yelich). CRC Press LLC, Boca Raton, FL, USA. pp. 287-295.

Barrios, B., R. Perez-Pe, M. Gallego, A. Tato, J. Osada, T. Muino-Blanco, and J. A. Cebrian-Perez. 2000. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol. Reprod. 63:1531-1537.

Buege, J. A. and S. D. Aust. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310.

Chaveiro, A., L. Machado, A. Frijters, B. Engel, and H. Woelders. 2006. Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology 65:1875-1890.

Cheema, R. S., V Zodinsanga, and A. K. Bansal. 2015. Characterization and immunolocalization of HBP, FA-1 and TIMP-2 like proteins in cattle bull semen: HBP modification in in vitro capacitated spermatozoa. Theriogenol. Insight 5:53-67.

Farooqui, A. A. 1980. Purification of enzymes by heparinsepharose affinity chromatography. J. Chromatogr. A. 184:335-345.

Fraser, L. R., L. R. Abeydeera, and K. Niwa. 1995. [Ca.sup.2+]-Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis. Mol. Reprod. Dev. 40:233-241.

Harshan, H. M., L. P. Singh, A. Arangasamy, M. R. Ansari, and S. Umar. 2006. Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa. Anim. Reprod. Sci. 93:124-133.

Jeyendran, R. S., H. H. Vander-Ven, M. Perez-Pelaez, B. G. Crabo, and L. J. D. Zanevld. 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Repord. Fertil. 70: 219-228.

Karunakaran, M., T. G. Devanathan, K. Kulasekar, P. Sridevi, T. Jawahar, K. Loganatahsamy, A. Dhali, and S. Selvaraju. 2012. Effect of fertility associated protein on oxidative Stress of bovine sperm cells. Ind. J. Anim. Reprod. 33:43-47.

Kumar, A., L. P. Singh, H. M. Harshan, and A. C. Majumdar. 2008. Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP). Anim. Reprod. Sci. 104:220-226.

Lessard, C., S. Parent, P. Leclerc, J. L. Bailey, and R. Sullivan. 2000. Cryopreservation alters the levels of the bull sperm surface protein P25b. J. Androl. 21:700-707.

Lowry, O. H., N. J. Rosenberg, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.

Manaskova, P., J. Liberda, M. Ticha, and V Jonakova. 2002. Isolation of non-heparin-binding and heparin-binding proteins of boar prostate. J. Chromatograph. B. 770:137-143.

Manjunath P, V Nauc, A. Bergeron, and M. Me'nard. 2002. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk. Biol. Reprod. 67:1250-1258.

Miller, D. J., M. A. Winer, and R. L. Ax. 1990. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod. 42:899-915.

Mogielnicka-Brzozowska, M. and W. Kordan. 2011. Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals. Pol. J. Vet. Sci. 14:489-499.

Mogielnicka-Brzozowska, M., P. Wysocki, J. Strzezek, and W. Kordan. 2011. Zinc-binding proteins from boar seminal plasma --isolation, biochemical characteristics and influence on spermatozoa stored at 4[degrees]C. Acta Biochim. Pol. 58:171-177.

Mota Filho, A. C., H. V Silva, T. G. Nunes, M. B. de Souza, L. A. de Freitas, A. A. de Araujo, and L. D. M. da Silva. 2014. Cryopreservation of canine epididymal sperm using ACP-106c and TRIS. Cryobiology 69:17-21.

Nass, S. J., D. J. Miller, M. A. Winer, and R. L. Ax. 1990. Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent. Mol. Reprod. Dev. 25:237-246.

Olaciregui, M. L. Gil, A. Monton, V Luno, R. A. Jerez, and J. I. Marti. 2014. Cryopreservation of epididymal stallion sperm. Cryobiology 68:91-95.

Raval, R. J. and A. J. Dhami. 2010. Effect of additives on various spermatozoal attributes of fresh, frozen-thawed and refrigerated semen. Ind. J. Anim. Reprod. 31:33-36.

Sharma, R. K. and A. Agarwal. 1996. Role of reactive oxygen species in male infertility. Urology 48:835-850.

Shi, Y, D. D. Mosser, and R. I. Morimoto. 1998. Molecularchaperones as HSF1 -specific transcriptional repressors. Genes Dev. 12:654-666.

Sidhu, R. S., R. K.Sharma, A. J. Jr. Thomas, and A. Agarwal. 1998. Relationship between creatine kinase activity and semen characteristics in subfertile men. Int. J. Fertil. Women's. Med. 43:192-197.

Srivastava, N., S. K. Srivastava, S. K. Ghosh, A. Kumar, P. Perumal, and A. Jerome. 2013. Acrosome membrane integrity and cryocapacitation are related to cholesterol content of bull spermatozoa. Asian Pac. J. Reprod. 2:126-131.

Therien, I., D. Bousquet, and P. Manjunath. 2001. Effect of seminal phospholipid-binding proteins and follicular fluid on bovine sperm capacitation. Biol. Reprod. 65:41-51.

Therien, I., R. Moreau, and P. Manjunath. 1998. Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm. Biol. Reprod. 59:768-776.

Tvrda, E., Z. Knazicka, L. Bardos, P. Massanyi, and N. Lukac. 2011. Impact of oxidative stress on male fertility--A review. Acta Vet. Hung. 59:465-484.

Ward, C. R. and B. T. Storey. 1984. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol. 104:287-296.

Watson, P. F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 61:481-492.

Yoon, S. J., W. S. Kwon, M. S. Rahman, J. S. Lee, and M. G. Pang. 2015. A novel approach to identifying physical markers of cryo-damage in bull spermatozoa. PLoS One. 10:e0126232.

Maulikkumar Patel, Vinod K. Gandotra, Ranjna S. Cheema *, Amrit K. Bansal, and Ajeet Kumar

Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Science University (GADVASU), Ludhiana, Punjab 141004, India

* Corresponding Author: Ranjna S. Cheema. Tel: +91-94644 16085, E-mail: ranjna.cheema@gmail.com

Submitted Jul. 10, 2015; Revised Aug. 18, 2015; Accepted Sept. 11, 2015

Table 1. Effect of different doses of affinity purified SP-HBP on
sperm function and fertility tests during pre-freeze and frozen-thaw
phases of cryopreservation

Sperm parameter                                Pre-freeze

                              C (1)                     10

Motility (%)          73.3 [+ or -] 1.7 (a)    76.7 [+ or -] 1.7 (a)
Viability (%)         68.7 [+ or -] 3.6 (ab)   61.2 [+ or -] 1.2 (a)
HOST (%)              35.3 [+ or -] 2.4 (a)    38.2 [+ or -] 4.3 (a)
Acrosome damage (%)
  Cryo-Cap            22.9 [+ or -] 2.6 (a)    22.1 [+ or -] 1.6 (a)
  Cryo-AR             12.1 [+ or -] 4.5 (a)    10.1 [+ or -] 3.8 (a)

In vitro capacitation/acrosome reaction (%)
  Cap                           --                      --
  AR                            --                      --
  LPO                 14.7 [+ or -] 5.7 (a)    12.4 [+ or -] 3.8 (a)
  (MDA, [micro]M/
    [10.sup.9]
    spermatozoa)

Sperm parameter                                Pre-freeze

                                20                       40

Motility (%)          83.3 [+ or -] 1.7 (b)    75.0 [+ or -] 0.0 (a)
Viability (%)         76.2 [+ or -] 3.5 (ab)   72.4 [+ or -] 3.1 (b)
HOST (%)              42.1 [+ or -] 4.1 (a)    35.72 [+ or -] 2.2 (a)
Acrosome damage (%)
  Cryo-Cap            23.3 [+ or -] 1.5 (a)    24.5 [+ or -] 1.8 (a)
  Cryo-AR             10.5 [+ or -] 4.4 (a)    11.5 [+ or -] 4.9 (a)

In vitro capacitation/acrosome reaction (%)
  Cap                           --                       --
  AR                            --                       --
  LPO                 11.9 [+ or -] 3.6 (a)    14.5 [+ or -] 4.6 (a)
  (MDA, [micro]M/
    [10.sup.9]
    spermatozoa)

Sperm parameter                               Frozen-thaw

                                C                       10

Motility (%)          45.0 [+ or -] 2.9 (c)   46.7 [+ or -] 1.7 (c)
Viability (%)         48.7 [+ or -] 8.7 (c)   47.8 [+ or -] 6.2 (c)
HOST (%)              35.0 [+ or -] 2.2 (a)   12.1 [+ or -] 2.4 (b)
Acrosome damage (%)
  Cryo-Cap            29.2 [+ or -] 2.9 (b)   28.1 [+ or -] 3.4 (b)
  Cryo-AR             16.3 [+ or -] 4.3 (a)   13.8 [+ or -] 2.8 (a)

In vitro capacitation/acrosome reaction (%)
  Cap                 39.1 [+ or -] 3.4 (a)   39.4 [+ or -] 2.8 (a)
  AR                  27.9 [+ or -] 5.8 (a)   30.1 [+ or -] 6.1 (ab)
  LPO                 50.3 [+ or -] 2.8 (b)   44.3 [+ or -] 4.1 (bc)
  (MDA, [micro]M/
    [10.sup.9]
    spermatozoa)

Sperm parameter                               Frozen-thaw

                               20                       40

Motility (%)           58.3 [+ or -] 4.4d     48.3 [+ or -] 4.4 (c)
Viability (%)         56.0 [+ or -] 7.6 (c)   52.4 [+ or -] 8.2 (c)
HOST (%)              18.6 [+ or -] 3.9 (b)   14.4 [+ or -] 3.0 (b)
Acrosome damage (%)
  Cryo-Cap            28.4 [+ or -] 1.7 (b)   30.1 [+ or -] 2.4 (b)
  Cryo-AR             14.6 [+ or -] 3.7 (a)   17.0 [+ or -] 4.2 (a)

In vitro capacitation/acrosome reaction (%)
  Cap                 44.4 [+ or -] 3.0 (a)   42.5 [+ or -] 3.2 (a)
  AR                  34.0 [+ or -] 5.1 (b)   33.4 [+ or -] 5.0 (b)
  LPO                 40.3 [+ or -] 2.6 (c)   45.3 [+ or -] 4.9 (bc)
  (MDA, [micro]M/
    [10.sup.9]
    spermatozoa)

HOST, hypo-osmotic swelling test; Cap, capacitated; AR, acrosome
reacted; LPO, lipid peroxidation; MDA, malondialdehyde.

(1) C, control (0), 10, 20, 40 (Dose of HBP in gg/mL).

Superscripts (a, b, ab, bc) indicate the difference at 5% level of
significance within the rows.

Table 2. Effect of affinity purified SP-HBP on sperm motility,
viability and HOST (mean [+ or -] SE) during pre-freeze and frozen-
thaw phases of cryopreservation

                            Motility (%)

Bull number                 Pre-freeze

                     C1                  T1

1             70.0 [+ or -] 0.0   80.0 [+ or -] 0.0
2             70.0 [+ or -] 2.9   78.3 [+ or -] 3.3
3             68.3 [+ or -] 4.4   73.3 [+ or -] 4.4
4             61.7 [+ or -] 1.7   70.0 [+ or -] 2.9
5             63.3 [+ or -] 3.3   68.3 [+ or -] 3.3
6             65.0 [+ or -] 2.9   71.7 [+ or -] 1.7
Combination       66.4 (a)            73.6  (b)
factor mean

                            Motility (%)

Bull number                 Frozen-thawed

                      C                   T

1             58.3 [+ or -] 3.4   66.6 [+ or -] 2.9
2             55.0 [+ or -] 2.9   61.7 [+ or -] 1.7
3             50.0 [+ or -] 5.8   56.6 [+ or -] 4.4
4             45.0 [+ or -] 2.9   50.0 [+ or -] 2.9
5             41.7 [+ or -] 4.4   43.3 [+ or -] 3.3
6             46.7 [+ or -] 6.0   51.7 [+ or -] 6.0
Combination       49.4 (c)              55.0d
factor mean

                            Viability

Bull number                 Pre-freeze

                      C                   T

1             73.0 [+ or -] 6.6   79.6 [+ or -] 4.5
2             69.1 [+ or -] 7.5   76.9 [+ or -] 5.8
3             65.9 [+ or -] 3.8   71.0 [+ or -] 3.6
4             61.3 [+ or -] 2.1   67.0 [+ or -] 1.0
5             55.7 [+ or -] 2.8   59.3 [+ or -] 3.3
6             65.1 [+ or -] 0.5   69.3 [+ or -] 1.8
Combination       65.0 (a)            70.5 (a)
factor mean

                            Viability

Bull number                 Frozen-thawed

                      C                   T

1             59.5 [+ or -] 2.2   64.8 [+ or -] 2.7
2             54.9 [+ or -] 7.1   61.6 [+ or -] 6.0
3             50.4 [+ or -] 8.8   56.4 [+ or -] 6.1
4             43.0 [+ or -] 7.2   47.2 [+ or -] 7.2
5             38.2 [+ or -] 3.1   44.9 [+ or -] 0.2
6             48.5 [+ or -] 1.4   59.5 [+ or -] 0.7
Combination       49.1 (b)            55.7 (b)
factor mean

                              HOST

Bull number                 Pre-freeze

                      C                   T

1             30.6 [+ or -] 3.7   36.2 [+ or -] 3.3
2             32.5 [+ or -] 8.1   54.6 [+ or -] 2.5
3             36.1 [+ or -] 8.0   46.3 [+ or -] 6.2
4             25.3 [+ or -] 5.1   30.1 [+ or -] 3.3
5             16.0 [+ or -] 0.7   22.0 [+ or -] 2.6
6             21.6 [+ or -] 4.6   32.5 [+ or -] 4.0
Combination       27.0 (a)            36.9 (b)
factor mean

                              HOST

Bull number                 Frozen-thawed

                      C                   T

1             18.1 [+ or -] 3.2   25.1 [+ or -] 4.9
2             16.9 [+ or -] 5.9   27.8 [+ or -] 4.8
3             27.5 [+ or -] 7.5   35.3 [+ or -] 5.7
4             17.7 [+ or -] 1.7   20.7 [+ or -] 2.1
5             8.8 [+ or -] 4.6    12.6 [+ or -] 3.2
6             12.6 [+ or -] 2.0   24.6 [+ or -] 1.5
Combination       16.9 (c)            24.3 (a)
factor mean

SP-HBP, seminal plasma-heparin binding protein; HOST, hypo-osmotic
swelling test; SE, standard error.

(1)  C, control without HBP; T, supplemented with SP-HBP.

Superscripts a, b, c, d indicate the difference at 5% level of
significance within the columns.

Table 3. Effect of affinity purified SP-HBP on acrosome damage (mean
[+ or -] SE) during pre-freeze and frozen-thaw phase of
cryopreservation

Bull number                    Pre-freeze

                               Control (1)

                 Capacitated      Acrosome reacted

1             20.2 [+ or -] 3.4   14.3 [+ or -] 4.1
2             25.1 [+ or -] 3.8    9.6 [+ or -] 2.1
3             20.1 [+ or -] 1.1    5.3 [+ or -] 0.3
4             22.9 [+ or -] 1.0    6.6 [+ or -] 0.1
5             30.6 [+ or -] 1.5    9.0 [+ or -] 0.4
6             21.5 [+ or -] 1.0   14.1 [+ or -] 2.1
Combination       23.4 (a)             9.8 (a)
factor mean

Bull number                    Pre-freeze

                               Treated (1)

                 Capacitated      Acrosome reacted

1             21.2 [+ or -] 3.6   11.7 [+ or -] 3.7
2             25.2 [+ or -] 3.7    8.5 [+ or -] 2.8
3             20.3 [+ or -] 0.8    4.9 [+ or -] 0.6
4             24.4 [+ or -] 0.7    5.2 [+ or -] 0.1
5             29.6 [+ or -] 0.9   10.8 [+ or -] 0.7
6             22.1 [+ or -] 0.5   12.2 [+ or -] 2.6
Combination       23.08 (a)            8.8 (a)
factor mean

Bull number                    Frozen-thawed

                               Control

                 Capacitated      Acrosome reacted

1             25.6 [+ or -] 4.5   16.8 [+ or -] 3.6
2             30.8 [+ or -] 3.3   13.3 [+ or -] 2.0
3             26.1 [+ or -] 1.4   10.9 [+ or -] 1.3
4             32.9 [+ or -] 0.8   14.0 [+ or -] 3.1
5             37.5 [+ or -] 0.7   13.5 [+ or -] 2.0
6             29.5 [+ or -] 0.6   21.1 [+ or -] 0.5
Combination       30.4 (b)            14.9 (bc)
factor mean

Bull number                    Frozen-thawed

                               Treated

                 Capacitated      Acrosome reacted

1             23.6 [+ or -] 3.4   15.8 [+ or -] 3.5
2             21.3 [+ or -] 0.8    4.9 [+ or -] 0.6
3             26.3 [+ or -] 1.4   10.1 [+ or -] 0.8
4             33.9 [+ or -] 0.4   12.4 [+ or -] 2.6
5             35.0 [+ or -] 1.2   15.0 [+ or -] 2.0
6             29.6 [+ or -] 0.2   19.8 [+ or -] 1.4
Combination       28.2 (bc)           13.0 (c)
factor mean

SP-HBP, seminal plasma-heparin binding protein; SE, standard error.

(1) Control, without HBP); Treated, supplemented with HBP.

Superscripts a, b, bc, c indicates the difference at 5% level of
significance within column.

Table 4. Effect of affinity purified SP-HBP on in vitro
capacitation/acrosome reaction (mean [+ or -] SE) during pre-freeze
and frozen-thaw phase of cryopreservation

Bull number                Control

                         Capacitated          Acrosome
                                               reacted

1                     41.7 [+ or -] 1.6   28.3 [+ or -] 5.7
2                     40.9 [+ or -] 0.4   24.4 [+ or -] 2.2
3                     36.6 [+ or -] 2.1   23.9 [+ or -] 2.2
4                     45.6 [+ or -] 1.8   25.0 [+ or -] 3.3
5                     43.0 [+ or -] 0.4   27.0 [+ or -] 2.9
6                     40.0 [+ or -] 2.0   31.7 [+ or -] 1.7
Average [+ or -] SE       41.4 (a)            26.3 (b)

Bull number                Treated

                         Capacitated          Acrosome
                                               reacted

1                     44.1 [+ or -] 1.6   33.5 [+ or -] 5.4
2                     46.9 [+ or -] 0.6   26.6 [+ or -] 2.5
3                     41.9 [+ or -] 1.7   30.0 [+ or -] 2.5
4                     47.1 [+ or -] 0.5   30.4 [+ or -] 2.9
5                     48.5 [+ or -] 0.2   27.7 [+ or -] 2.1
6                     44.3 [+ or -] 0.5   32.9 [+ or -] 3.7
Average [+ or -] SE       45.5 (c)            30.2 (d)

SP-HBP, seminal plasma-heparin binding protein; SE, standard error.

(1) Control, without HBP; Treated, supplemented with SP-HBP.

Superscripts a, b, c, d indicate the difference at 5% level of
significance within the columns.

Table 5. Effect of affinity purified SP-HBP on MDA production
([micro]M-[10.sup.9] spermatozoa, mean [+ or -] SE) during pre-
freeze and frozen-thaw phase of cryopreservation

Bull                            Pre-freeze
number
                 Control (1)          Treated (1)

1             73.5 [+ or -] 51.3   51.6 [+ or -] 35.0
2             46.8 [+ or -] 19.4   24.9 [+ or -] 12.8
3             22.1 [+ or -] 10.3   50.2 [+ or -] 33.4
4             72.9 [+ or -] 20.8   32.8 [+ or -] 5.4
5             97.0 [+ or -] 24.5   62.3 [+ or -] 22.6
6             51.6 [+ or -] 20.7   45.8 [+ or -] 20.9
Combination       66.7 (ac)             44.6 (a)
factor mean

Bull                             Frozen-thaw
number
                    Control               Treated

1             112.4 [+ or -] 38.0   94.9 [+ or -] 37.8
2              83.3 [+ or -] 19.4   60.3 [+ or -] 15.1
3             115.2 [+ or -] 34.1   98.9 [+ or -] 31.6
4             130.3 [+ or -] 47.5   65.6 [+ or -] 16.8
5             132.8 [+ or -] 49.6   85.4 [+ or -] 31.5
6             159.7 [+ or -] 69.9   132.7 [+ or -] 51.1
Combination       122.3 (bd)             89.6 (cd)
factor mean

SP-HBP, seminal plasma-heparin binding protein; MDA,
malondialdehyde; SE, standard error.

(1) Control, without HBP; Treated, supplemented with SP-HBP.

Superscripts a, ac, bd, cd indicates the difference at 5% level of
significance within the colums.


----------

Please note: Some tables or figures were omitted from this article.
COPYRIGHT 2016 Asian - Australasian Association of Animal Production Societies
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2016 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Patel, Maulikkumar; Gandotra, Vinod K.; Cheema, Ranjna S.; Bansal, Amrit K.; Kumar, Ajeet
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Sep 1, 2016
Words:6927
Previous Article:Comparative proteomic analysis of yak follicular fluid during estrus.
Next Article:Novel polymorphisms of adrenergic, alpha-1B-, receptor and peroxisome proliferator-activated receptor gamma, coactivator 1 beta genes and their...
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters