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Philippine Journal of Science144 (2): 209-219, December 2015ISSN 0031 - 7683Date Received: ?? Feb 20??

Key words: Buffalo, fertility, HOST, spermatozoa, sperm membrane integrity, semen

Hypo-Osmotic Swelling Test for Membrane Integrity Evaluation of Frozen-Thawed Water Buffalo

(Bubalus bubalis Linn.) Spermatozoa

1Reproductive Biotechnology Laboratory, Philippine Carabao Center, Science City of Muñoz, Nueva Ecija, Philippines.

2Department of Biological Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija, Philippines

3Prometeo, Universidad Tecnica de Babahoyo, Los Rios, Ecuador

*Corresponding author: [email protected] [email protected]

Danilda Hufana-Duran1,2,3*, Rachelle P. Mallari2, Devon P. Suba2, Peregrino G. Duran1, Evaristo A. Abella2, and Felomino V. Mamuad1

Studies were conducted to establish the standard procedure of hypo-osmotic swelling test for frozen-thawed water buffalo spermatozoa. Hypo-osmotic swelling test is used in assessing plasma membrane integrity of spermatozoa of human and other livestock species. Plasma membrane integrity is a requisite of a fertile sperm. Standardization of hypo-osmotic swelling solutions and methodologies has not yet been done for frozen-thawed water buffalo spermatozoa; thus, the objective of this study was to identify assay conditions specifically for water buffalo. The following studies were conducted: Effective exposure time (0, 15, 30, 45, 60, 75, 90, 105, and 120 min) and temperature (25° C or 37° C), examination of the best osmolality of solution (0, 50, 100, 150, 200, 250, and 300 mOsm), evaluation of sugar (fructose vs. sucrose) better for HOS solution, and assessment of the repeatability and consistency of the test. Results showed optimum sperm reaction at 150 mOsm at 60 min but no significant difference was observed to 15 to 60 min exposure at 37° C in 50 to 200 mOsm using either fructose or sucrose as hypo-osmotic swelling solutions. Using the optimum HOST treatment procedure using 150 mOsm fructose-sodium citrate solution at 37° C for 45 min, a coefficient of variation ranging from 0.05 to 0.09 among bulls was observed. Results demonstrated a standard hypo-osmotic swelling test procedure that is simple, accurate, and consistent with good repeatability to predict the functional membrane integrity of buffalo spermatozoa.

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02:23 P.M.

INTRODUCTIONWater buffalo (Bubalus bubalis Linn.) is known for its inferior reproductive potential compared to cattle. In carrying out artificial insemination (AI) and in vitro fertilization (IVF), the efficiency rate is lower in water buffalo compared to cattle and the quality of spermatozoa significantly affects the success rate (Hufana-Duran et

al. 2005). Though frozen semen used for AI and IVF undergoes rigorous semen quality evaluation prior to processing, the post-thaw quality needs to be reassessed to ensure that the sperm processing, storage, and transport processes have not affected the quality of the semen. Assessment of sperm quality post-thawing is usually done by motility analysis which is helpful but very subjective and not sufficient to provide the quality picture of semen. It has been indicated that sperm morphology, sperm concentration, and sperm motility are the three

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major components of routine sperm quality assessment (Sutkeviciene et al. 2009) and are inexpensive. But these parameters are subjective and are indicated to be of low predictive power to monitor testicular function of boars and bulls since spermatozoa are not analyzed in terms of their total integrity (Soderquist et al. 1991; Januskauskas et al. 2001; Andrade et al. 2007). Furthermore, recent findings (Bassiri et al. 2012) showed that sperm cells assessed by motility and morphology still possess DNA damage. In fact, Avendaño et al. (2009) estimated that one out of every two oocytes is likely to be inseminated by a spermatozoon that has damaged DNA. Similarly, computer-assisted analysis of spermatozoa which provides rapid and objective evaluation of the semen quality requires special instruments and software which are expensive and sometimes not readily available in an average breeding farm and laboratory. Prediction of potential fertility of the spermatozoa is an important consideration to ensure high efficiency in AI and IVF and doing this on the basis of single assay is not reliable (Lodhi et al. 2008). Therefore, looking for other techniques that may be inexpensive, objective, and easily performed without the assistance of expensive, sophisticated instruments becomes imperative.

Sperm membrane is necessary in maintaining both the biochemical and structural integrity of spermatozoon (Cabrita et al. 1999) and membrane integrity is of fundamental importance in assessing the fertilizing capacity (Jeyendran et al. 1984). Plasma membrane integrity of sperm is of crucial importance for optimal sperm function (Khan & Ijaz 2008) because only a sperm with an intact plasma membrane can undergo a series of complex changes in the female reproductive tract and acquire the ability to fertilize an oocyte (Yanagimachi 1994). Therefore, plasma membrane is one of the most relevant spermatological parameters in assessing semen quality.

The hypo-osmotic swelling test (HOST) for spermatozoa is a test that indicates the functional integrity of the sperm membrane (Mandal et al. 2003). It involves exposure of the spermatozoon to hypo-osmotic solutions where biochemically-active spermatozoa increase their volume in order to establish equilibrium between the fluid compartment within the spermatozoa and the extracellular environment. Both cell size and shape of the spermatozoon changes and can be seen as swelling by a phase contrast microscope (Cabrita et al. 1999). This swelling process culminates in promoting spherical expansion of the cell membrane covering the tail, thus forcing the flagellum to coil inside the membrane. Coiling of the tail begins at the distal end and proceeds towards the midpiece and head as the osmotic pressure of the suspending media is lowered (Jeyendran et al. 1984). This test has been developed and used in human (Jeyendran et al. 1984) and later confirmed as a good tool for evaluating the sperm membrane integrity

of various species: canine (Rodriguez et al. 1994), rainbow trout (Cabrita et al. 1999), bovine (Rota et al. 2000), equine (Neild et al. 2000), porcine (Perez-Llano et al. 2001), and rabbit (Amorin et al. 2009), with few trials in water buffalo (Mandal et al. 2003, Lodhi et al. 2008; Iqbal et al. 2009).

Although HOST assay has been standardized successfully in mouse (Sliwa 1993), canine (Pinto et al. 2008), bovine (Correa et al. 1997), human (Jeyendran et al. 1984), stallion (Nie & Wenzel 2001), goat (Fonseca et al. 2005), and pig (Samardzija et al. 2008), standard procedure of this technique specifically for buffalo semen is lacking. Application of this assay technique on buffalo (Mandal et al. 2003; Lodhi et al. 2008; Iqbal et al. 2009) have been adopted from the procedures developed from other species. As a result, Lodhi et al. (2008) achieved a significant (P<0.05) positive correlation between progressive motility, morphologically normal spermatozoa, sperm viability, and percentage of HOST positive spermatozoa but Iqbal et al. (2009) achieved a weak relationship between sperm viability and HOST (r=-0.12). The effect of the adopted procedures on water buffalo sperm cells might hamper the accuracy of the reported data. As noted, Lodhi et al. (2008) used 150 mOsm/L while Iqbal et al. (2009) used 190 mOsm/L sodium citrate fructose solutions in treating fresh semen of Nili Ravi bulls. Though in human 190 mOsm/L (Jeyendran et al. 1984; Tartagni et al. 2002) is used, 125 mOsm/L solutions is the best for fresh goat spermatozoa (Fonseca et al. 2005) compared with pig which is 100 mOsm/L (Samardzija et al. 2008), indicating species differences. Moreover, HOS Test solutions and methodologies have not been evaluated specifically for assessing water buffalo spermatozoa; hence, the objective of this study was to identify a HOST assay conditions specifically for frozen-thawed water buffalo sperm cells that would maximize the spermatozoal plasma membrane swelling. The study is concerned with establishing the best condition for HOS Test based on exposure time and incubation temperature, osmolality of solution, and effective solution for HOS Test in frozen-thawed water buffalo sperm cells, and assess the repeatability of the assay procedure.

MATERIALS AND METHODS

Frozen Semen and EvaluationFrozen semen processed by the Semen Processing Laboratory of the Philippine Carabao Center in Carranglan, Nueva Ecija, Philippines were used in the studies. In Study 1 to 3, frozen semen from three bulls intended for IVF was pooled and used. For each treatment comparison, frozen semen sample from the same pooled semen straw was

Hufana Duran et al.: Hypo-Osmotic Swelling Test for Membrane Integrity Evaluation of Frozen-Thawed Water Buffalo

Philippine Journal of ScienceVol. 144 No. 2, December 2015

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Figure 1. Schematic presentation of no reaction (a) and with mild (b) and severe (c-g) HOST reaction sperm cells. Adopted from the criteria described by Jeyendran et al. (1984) and Fonseca et al. (2005).

utilized. In Study 4, frozen semen from three individual bulls was used and all replications were made using frozen semen sample from the same ejaculate. All microscopic evaluations were made and reviewed by three staff.

General Procedure Frozen semen was thawed at 39° C for 15 s, layered in 15 mL centrifuge tube containing 39° C pre-warmed Brackett and Oliphant (Brackett & Oliphant 1975) medium (BO medium) and centrifuged at 1,500 rpm for 5 min to wash the sperm cells. The supernatant was discarded leaving the sperm pellet. The sperm pellet was disturbed to mix well. For every semen straw sample thawed and washed with BO medium, a sample was taken for sperm tail pre-examination to determine the baseline data of any tail swelling as described by WHO (1999). The data obtained in the pre-examination was deducted from the data after HOS Test analysis. The remaining sperm pellet was then subjected to various treatments.

Of the dense sperm sample after centrifugation and removal of supernatant, twenty microliter was taken and was added to 80µL of HOST solution in a 1.5 mL microcentrifuge tube. For the HOST analysis, a 10 µL drop of sample was placed on a clean glass slide and was covered with cover slip. Slides were examined at x400 using phase contrast microscope. A total of 200 spermatozoa were observed for changes associated with swelling based on the appearance of swelled sperm cells described by Jeyendran et al. (1984) and Foncesca et al. (2005) taking notes on the abnormalities described by Barth and Oko

et al. (1989) that were avoided in the counting. Sperm noted to be abnormal were not included in the gathered data of swelled sperm. The percentage of HOST-reacted spermatozoa i.e. number of spermatozoa with coiled tails per total number of spermatozoa examined x100 was recorded for each sample. Figure 1 presents the appearance of HOST reactive sperm cells.

To confirm the effect of exposure of the water buffalo spermatozoa to HOS Test, the livability was examined using the eosin-nigrosin staining technique. Microscope slides and nigrosin-eosin stain are pre-warmed to body temperature then a 5 µL drop of stain is pipetted onto the end of a slide followed by a 5 µL droplet of semen and loaded next to the stain. Another slide glass is placed on the edge of the drops of stain and semen and rocked back and forth a few times to mix the sperm and stain, and then smeared the second slide across the surface of the first. The slides are dried rapidly by placing in on a warming plate or waving it back and forth in the air. Smears are examined using a bright field microscope (40x objective lens) and normal live sperm that exclude the eosin stain and appear white in color was classified as live; whereas, sperm (i.e. those with loss of membrane integrity) that absorbed the eosin and appear pinkish in color was considered "dead". In each treatment replicate, a total of 100 sperm cells are counted and the number of live and dead sperm cells is recorded.

Experimental DesignTo develop the HOST for water buffalo, four studies were carried out: Study 1 tested the effect of exposure time (0,



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Table 1. Proportion of sugar and salt in the preparation of the different HOST solutions at 10 mL volume.

mOsm/L 0 50 100 150 200 250 300

Na citrate, g 0 0.0245 0.0490 0.0735 0.0980 0.1225 0.1470

Fructose, g 0 0.0450 0.0901 0.1351 0.1802 0.2252 0.2702

Molecular weights: sodium citrate, 294.1; fructose, 180.16

15, 30, 45, 60, 75, 90, 105, and 120 min) and temperature (25° C or 37° C); Study 2 examined the osmolality of solution (0, 50, 100, 150, 200, 250, and 300 mOsm/L); Study 3 assessed which HOST solution (fructose vs. sucrose in sodium citrate) is better, and; Study 4 assessed the repeatability of the test.

Effect of exposure time and temperatureTo check the effect of exposure time and temperature, frozen-thawed semen exposed to same HOST solution (150 mOsmol/L fructose and sodium citrate) were subjected to body temperature (37° C in a water bath) or in room temperature (26 to 28°C) then swelling reaction of sperm cells were examined at 0 (upon exposure), 15, 30, 45, 60, 75, 90, 105, 120 min. Swelling reaction was categorized as mild or severe depending on the appearance of the sperm cell based on the schematic presentation described by Jeyendran et al. (1984): (1) Mild if the sperm reacted with a small enlargement at the tip of the tail cause by swelling as seen in sperm image b. (2) Severe if the tail bent, or a hairpin curvature appeared, or the tail shortened and thickened, or a swollen area appeared that partly or completely enveloped the curved tail of the spermatozoon like sperm images c to g (Figure 1). A total of 200 sperm cells were examined for each slides of 5 replications.

Effect of osmolality of the solution To determine the effect of osmolality of the solution, sperm cells were exposed to 0, 50, 100, 150, 200, 250, and 300 mOsmol of fructose and sodium citrate solution. Preparation of the combination of sugar and salt solution was conducted by mixing the desired proportions of sugar and salt as described in Table 1. Swelling reaction was examined at temperature (37°C) and exposure time (45 min) identified best in Study 1. Preparation of the different HOST solutions is presented in Table 1.

Effect of sucrose vs. fructose in combination with sodium citrate as HOST solution Two different sugar and salt combinations: (sucrose and sodium citrate and fructose and sodium citrate) were assessed according to their ability to promote swelling on the spermatozoal plasma membrane. Using the best osmolality of the solution (150 mOsm) determined in

Study 2 and best incubation temperature (37° C) and exposure time (45 min) determined in Study 1, the sperm cells were exposed to two treatments: sucrose vs. fructose in combination with sodium citrate and the HOST reaction was evaluated.

Repeatability of HOSTTo check the repeatability of the HOST, frozen semen from 3 bulls were used and subjected to the HOST examination following the best exposure time (45 min), incubation temperature (37° C), osmolality (150 mOsm), and sugar-salt solution (fructose and sodium citrate) identified in Study 1 to 3. The test was replicated 5 times using single straw of frozen-thawed semen to check the repeatability of the results.

Statistical AnalysisResults were expressed in averages and percentages, and standard deviations were computed. Repeated measures ANOVA was used to test the main effects of exposure time, incubation temperatures, osmolality, and HOST solutions. Tukey's multiple comparison tests was used to compare means and determine the statistical difference between treatment groups. The repeatability was calculated from Analysis of Variance using Excel’s Single Factor Analysis ToolPak.

RESULTS AND DISCCUSSIONS

Exposure Time and TemperatureTable 2 presents the results in swelling reaction as affected by the incubation temperature. Figure 2 shows the HOST reacted and non-reacted sperm cells. Immediate severe reaction (P<0.05) was observed when sperm cells were exposed to HOST solution at Room Temperature (26-28º C) but not in Water Bath at 37° C (P<0.01), evidenced by an average reaction of 43.5% upon exposure (0 min) at 37° C against 67.2% at Room Temperature. An optimum reaction (71.7%) was noted in 15 min of exposure up to 90 min with slight decrease. However, this was not significantly different to 120 min. The results indicated that osmosis in sperm plasma membrane is affected by temperature, with room temperature of 26-28º C enhancing immediate HOST reaction while 37° C gives the optimum reaction at longer exposure time. The results also suggests that 15 to 120 min exposure does not significantly alter the results of the hypo-osmotic swelling test demonstrating that once the treated sperm cells swelled, the condition remain consistent up to 90 min. A decrease in coiled sperm was observed after 90 min of HOS exposure and this was consistent both at Room Temperature (26-28º C) and Water Bath (37° C) storage



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Table 2. Percent swelled water buffalo (Bubalus bubalis Linn.) sperm cells after exposure to hypoosmotic solution at different exposure time and incubation temperature.

Exposure time, min

Room temperature26-28°C, %±SE

Water bath37°C, %±SE

Mild Severe TOTAL Mild Severe TOTAL

0 5.0±0.0 62.2±0.1 67.2±0.1A 8.7±0.0a 34.8±0.1a 43.5±0.1aB

15 10.0±0.1 51.2±0.1 61.2±0.1A 9.5±0.0a 62.2±0.1b 71.7±0.1bB

30 4.8±0.1 60.8±0.2 65.6±0.2A 11.0±0.0a 56.7±0.1b 67.7±0.1bA

45 11.2±0.1 52.0±0.1 63.2±0.2A 8.2±0.0a 64.7±0.2b 72.9±0.2bB

60 12.2±0.1 50.0±0.1 62.2±0.2A 17.0±0.1a 54.7±0.1b 71.7±0.2bB

75 7.0±0.0 56.3±0.1 63.3±0.1A 14.5±0.1a 56.0±0.1b 70.5±0.2bB

90 8.5±0.0 54.7±0.1 63.2±0.1A 20.2±0.1ab 50.6±0.0b 70.9±0.2bB

105 8.4±0.0 51.7±0.1 60.1±0.1A 14.0±0.0a 48.8±0.1ab 62.8±0.2bA

120 9.3±0.0 55.0±0.1 64.3±0.1A 31.0±0.1b 33.1±0.1a 64.0±0.2bA

Values with different lower and upper case superscript are significantly different, P<0.05. Lower case superscript denotes mean differences between exposure times. Upper case superscript denotes differences between incubation temperatures.

Figure 2. Images of HOST reacted (+) and HOST non-reacted (-) water buffalo spermatozoa.



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Figure 3. Percent live water buffalo (Bubalus bubalis Linn.) spermatozoa after exposure to 37° C and 25° C at various exposure time.

condition. This suggests that at longer period, membrane damage may have occurred resulting in leakage, thus, the coiled tail stretched as a response to an empty membrane. In fact, Smikle et al. (1997) indicated that prolonged hypo-osmotic stress may lead to irreversible effects on the cell membrane and cause cell death and this was established by Buckett (2003). This observation was confirmed by livability test presenting increased percentage of dead sperm after 90 min of exposure (Figure 3).

The observed positive effect of 37° C as incubation temperature for HOST was also observed in human (Jeyendran et al. 1984), bovine (Correa et al. 1997), in swine (Vazquez et al. 1997), and in fresh Nili Ravi buffalo semen (Zubair et al. 2013). However, human spermatozoa react to HOST within the first 30 min in cattle, within 5 min, in swine within 30 to 120 min exposure time, while, in water buffalo in the present research within 15 to 120 min. The study by Zubair et al. (2013) in Nili Ravi buffalo used a fix 1 hour incubation time, hence, cannot be compared with these findings. The observed differences in the timing of reaction of sperm cells in the HOS Test could be attributed to the initial treatment conditions that the frozen semen samples were subjected to during cryopreservation and thawing and the effect of species differences.

Assessment of sperm livability (Fig. 3) showed high percentage of live sperm cells at 37° C but exposure to 26 to 28° C resulted to high percentage of dead spermatozoa. The high percentage of dead sperm cells at 26-28° C may indicate the effect of temperature shock among treated

sperm cells which could be associated by abrupt swelling reaction as shown in Table 2. This might be caused by the thawing of the semen at 39° C then storing it to 26 to 28° C causing a leak on the sperm plasma membrane as evident in the penetration of the eosin-nigrosin stain. This can be supported by earlier reports indicating that cooling is a major stressor, the result of which is the reorientation of membrane bound phospholipids into a different configuration that disrupt membrane function and permeability (Amann and Graham 1993; Lessard et al. 2000). The sperm thawing at 39º C and carrying the HOS Test at room temperature (26-28º C) was not beneficial as it resulted to high percentage of dead spermatozoa due to the stress response by spermatozoa as a reaction to a drop in temperature. This is supported by earlier claim (Lemma 2011) indicating that generally, temperature shock damage manifests itself as a decline in cell metabolism, altered membrane permeability, loss of intracellular components, and an increase in the number of dead spermatozoa. Lemma (2011) also reported that damage to the cellular membranes is of most significant effect of temperature shock because it has a carry-over effect on other cellular structures and functions.

It can be deduced from the results that, at 37° C exposure, temperature did not incur any temperature shock as the percentage of live sperm after thawing at 39º C are just the same in sperm cells exposed to HOST at 37º C (data not shown). The results imply that spermatozoa reacted to HOST suddenly until the osmotic equilibrium was met as evidenced by the significantly lower HOST (+) at 0

Minutes



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Figure 4. Percentage of swelled water buffalo (Bubalus bubalis Linn.) sperm cells after treatment in different osmolality of HOST solution . P<0.05

min and increased optimum at 15 min, consistent to the rest of different exposure time. These results showed that once osmotic equilibrium is met at favorable temperature, the condition of the sperm cells will be consistent and maintained allowing observation of the functional integrity of the sperm cells. The same observation was observed in human (Jeyendran et al. 1984), goat (Fonseca et al. 2005), and rabbit (Amorin et al. 2009). However, prolonged exposure beyond 120 min could revert the results as membrane integrity can be affected by hypo-osmotic stress as earlier mentioned (Smikle & Turek 1997).

Osmolality of the SolutionWhen the effect of osmolality was assessed (Study 2), optimum reaction of sperm cells was observed at 50 to 200 mOsm with peak at 150 mOsm (range of 142 to 157 mOsm; mean = 149.5 mOsm) sodium citrate - fructose solution with 52.4% reactive sperm cells. In contrast, the least percent swelling was obtained at 300 mOsm (range of 299.33 to 301.33 mOsm ; mean = 300.33 mOsm) and 0 mOsm with 34.2% and 39.75% of HOST reactive sperm cells, respectively. They were found to be significantly different (P<0.05) to 150 mOsm but not to 50, 100, 200, and 250 mOsm. These results showed that 150 mOsm is the optimum osmolality of solution for HOST in water buffalo sperm cells. These finding supports the observations reported by Khan and Ijaz (2008) indicating that osmolality higher than the osmolality of the seminal fluid (256 mOsmol in NiliRavi buffalo) does not cause osmotic reaction to the spermatozoa. It can also be said

that the herein observed reaction in different osmolalities and the conflicting observations by Lodhi et al. (2008) and Iqbal et al. (2009) on the correlation of HOST to various semen quality parameters could be attributed to the individual difference and tolerance of sperm plasma membrane, the different osmotic pressure used, the type of semen and the differences on the processing technique. The results support earlier observations showing highest percentage of sperm tail coiling at 150 mOsm in fresh semen of Nili Ravi bulls (Lodhi et al. 2008), 100 -150 mOsm in boar sperm cells (Vasquez et al. 1997), and 125 mOsm in fresh goat spermatozoa (Fonseca et al. 2005).

The absence of substantial difference between 50 to 200 mOsm/L in the present experiment could be due to the use of cryopreserved seminal sample compared to the fresh semen used in the previous studies (Vazquez et al. 1997, Mandal et al. 2003; Fonseca et al. 2005; Lodhi et al. 2008; Khan & Ijaz 2008; Aromin et al. 2009; Iqbal et al. 2009). Generally, the mean percentage of HOST (+) sperm cells in the present study (44.09%) is lower than 85.25% of HOST reactive sperm cells of Nili Ravi bulls (Lodhi et al. 2008). The difference in the results could be due to the variation in the seminal sample used. Lodhi et al. (2008) used fresh semen while frozen semen was used in this study. Another reason could be the individual bull differences. Since HOST is a test for membrane function that indicates the fertilizing capacity of spermatozoa (Jeyendran et al. 1984), the over-all mean of HOST (+) spermatozoa revealed that the frozen-thawed water buffalo spermatozoa have lower functional membrane



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Table 4. Standard deviation, standard error and coefficient of variation in carrying out HOST in water buffalo (Bubalus bubalis Linn.) spermatozoa.

Bull Mean SD SE CV

1 41.33a 2.08 0.07 0.05

2 36.07b 3.9 0.11 0.09

3 26.00c 2.0 0.10 0.07

SE: Standard error of the mean, CV: Coefficient of varianceValues with different superscript are significantly different P<0.01

Table 3. Effect of sucrose and fructose in combination with sodium citrate as HOST solution on the swelling reaction of water buffalo (Bubalus bubalis Linn.) sperm cells.

HOST solutionSwelled,%±SD

HOS (+)Totalmild severe

sucrose 40.47±2.0a 21.73±3.0a 62.2±4.0

fructose 28.17±1.0b 34.16±2.0b 62.33±2.0

Values with different superscripts are significantly different (P<0.05).

integrity compared to fresh bull semen and this conforms with earlier observations (Lodhi et al. 2008; Khan & Ijaz 2008). This supports the fact that cryopreservation can decrease the fertilizing capacity/ fertility of the spermatozoa. Furthermore, the initial semen quality used in this experiment (Study 2, Table 3) was low, hence, a difference on biological reaction. Nonetheless, the results demonstrated a trend consistent to earlier observations (Vasquez et al. 1997; Fonseca et al. 2005; Lodhi et al. 2008) that HOST reaction is gained at 100 to 150 mOsm and the present results demonstrated that 150 mOsm is the optimum for HOST solution for water buffalo frozen-thawed sperm cells.

Choice of Sugar In Study 3, there was no significant difference in the overall percentage swelling among treated sperm cells when sucrose or fructose is used as sugar component of the HOST solutions (Table 3). However, a significant difference was observed on the severity of reaction where more (P<0.05) sperm cells severely reacted in fructose than in sucrose-based HOST solution. In other species, the sodium citrate-fructose HOST solution induced optimum swelling on fresh goat sperm cells (Fonseca et al. 2005), rams (Farshad et al. 2010), and human spermatozoa (Jeyendran et al. 1984) while sodium citrate-sucrose solution is best for stallion (Nie & Wenzel 2001; Mansour 2009) and fresh rabbit spermatozoa (Aromin et al. 2009). These observations may suggest that the choice of sugar is more of species difference. The variance observed on the severity of reaction among treated sperm cells in fructose-based HOS solution could be explained by the difference on the influx of water through the sperm membrane by the sucrose and fructose molecules that conforms to earlier observation in human sperm cells (Jeyendran et al. 1984). Fructose is a monosaccharide while sucrose is a disaccharide and these two sugars differ in size. While the osmolality of the solution is the same, the solute in fructose-based HOST solution differs from the sucrose-based solution due to the difference in the size of the molecule and molecular weight. This difference can cause changes in the influx of water into the membrane

which in turn can cause variation in the reaction of the sperm cells. With nearly the same percentage of swelled sperm for both solutions in the present study, the results suggest that either of these sugars could be used in examining the functional integrity of the sperm plasma membrane in water buffalo species. However, though the results suggest that water buffalo sperm cells are not critical on whether sucrose or fructose as the component of the HOST solution, the significant difference on the severity of response among treated spermatozoa may serve as reference that may have important impact on future research assessing DNA abnormalities on water buffalo sperm cells. Recent findings showed that the reaction of individual sperm cells in HOS Test is influenced by the degree of DNA abnormality (Stanger et al. 2008, 2010; Bassiri et al. 2012).

Conditions for and Repeatability of HOST for Water Buffalo Bull Semen Following the 45 min exposure time at 37°C in 150 mOsm fructose-sodium citrate solution, assessment of the repeatability of the HOST showed a coefficient of variation of 0.05, 0.09, and 0.07 for Bulls 1, 2, and 3, respectively, with no significant difference (Table 4). These results showed that HOS Test for water buffalo using 150 mOsm of fructose-sodium citrate solution at 37ºC for 45 min exposure time has a high repeatability demonstrating the reliability of the HOST assay procedure. This coefficient of variation is within the range compared to that of observed in human (Jeyendran et al. 1984) where coefficient of variation was 0.03 to 0.16 depending on ejaculate.

The mean HOST reaction among the three bulls were significantly (P<0.05) different. Bulls 1, 2, and 3 exhibited a HOST reaction of 41.33%, 36.07%, and 26.0% respectively (Table 4). These results suggest that HOST is an effective method to detect the individual differences among bull functional membrane integrity of sperm cells. Since functional membrane integrity is an indicator of the fertilizing capacity of the sperm cells (Jeyendran et al. 1984; Mandal et al. 2003), this test may be used to predict the fertility of bulls used for AI to ensure that bulls with high fertility will be used to optimize calf production.



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It was reported that the percentage of positive sperms to HOST varies with the fertility level (Jeyendran et al. 1984), individual bull (Prasad et al. 1999), mass activity, progressive motility, sperm count, total sperm with intact acrosome (Prasad et al. 1999) and season (Kale et al. 2000). Screening the semen donor bulls by this assay test will help the semen processing centers improve bull management and ensure that frozen semen production is of optimum quality.

CONCLUSIONS Based on the results, it can be concluded that the optimum treatment for HOS Test of water buffalo spermatozoa can be achieved using 150 mOsm fructose-sodium citrate solution at 37º C and exposure time of 45 min. The HOS Test is a friendly procedure as it can be carried out at osmolality of 50 to 150 mOsm using either sodium citrate-fructose or sodium citrate-sucrose HOST solution for 15 to 90 min exposure time without compromising the results. However, for better sperm reaction, fructose-based HOST solution is preferable. The assay technique has good repeatability, simple, and can be done within one and a half hour without jeopardizing the result of the analysis. Results of this study can serve as reference in the standard assay for the assessment of functional membrane integrity of water buffalo spermatozoa.

ACKNOWLEDGEMENTAuthors acknowledge the Semen Processing Laboratory of the Philippine Carabao Center (PCC) in Carranglaan, Nueva Ecija for the provision of frozen semen used in this study.

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