Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I

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Theriogenology 73 (2010) 1–10 www.theriojournal.com

Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I S. Selvaraju *, S. Nandi, T. Siva Subramani, B.S. Raghavendra, S.B.N. Rao, J.P. Ravindra Reproductive Physiology Laboratory, Animal Physiology Division, National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore 560030, India Received 28 November 2008; received in revised form 10 July 2009; accepted 12 July 2009

Abstract The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo (Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17  1.51) compared with that in the control group (9.50  0.36) at 2 h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30 min (33.27  2.62 vs. 26.71  1.02), 60 min (24.24  3.45 vs. 18.77  2.09), and 90 min (22.86  3.02 vs. 16.92  1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120 min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2 h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2 h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4 h (41.12  6.44 vs. 43.53  5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I–treated group (56.73  3.70) compared with that in the control group (44.85  2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility. # 2010 Elsevier Inc. All rights reserved. Keywords: IGF-I; Acrosin proteolytic activity; Acrosomal reaction; Mitochondrial membrane potential; Sperm-zona binding; Cleavage rate

1. Introduction Artificial insemination (AI) has become the most suitable and efficient method of breeding cattle and buffalo. However, conception rate after AI has been low and unpredictable. Causes for the reduced fertility are multifactorial ranging from inadequate management to * Corresponding author. Tel.: +91 80 25711304; fax: +91 80 25711420. E-mail address: [email protected] (S. Selvaraju). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.07.008

poor intrinsic gamete quality. The cooling, freezing, and thawing processes induce stress in the sperm membrane and reduce fertility [1]. In subfertile bulls, though spermatozoa appear to maintain adequate motility in vitro, the fertility is lower than expected under field condition. In these situations, improving the fertility potential of a semen sample becomes a necessity. Growth factors play an important role in the process of spermatogenesis, sperm motility, and fertility. Recent studies indicate the possible role of insulin-like growth factor-I (IGF-I) on sperm viability and motility in rats

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[2], cattle [3], and buffalo [4]. Insulin-like growth factor-I has an antioxidant effect and maintains sperm motility [4]. The testis secretes IGF-I [5], and the IGF-I receptors have been reported in the spermatogonia, spermatocytes, spermatids, and spermatozoa [3,6]. The prefertilization events in the female reproductive tract are regulated by an interaction between oviductal IGF-I and the IGF-I receptors on the spermatozoa [7,8]. Further, IGF-I concentrations in the seminal plasma of healthy breeding bulls/stallions were positively correlated with fertilization/pregnancy rates [9,10]. Hence, IGF-I has been suggested to be one of the important factors for germ cell development and maturation and the motility of the spermatozoa. Maintaining the molecular features of the sperm during the process of preservation, thawing, and transport in the female reproductive tract are essential for successful fertilization. In mammals, acrosin proteolytic activity is related to the fertilizing capacity of the spermatozoa [11,12]. Sperm penetration into zona pellucida is aided by hydrolytic enzymes in the sperm acrosome. Acrosin is a trypsin-like serine proteinase that is found exclusively within the acrosome of the mammalian spermatozoa [13,14]. Sperm acrosin proteolytic activity has been reported to influence in vitro fertility [12,15,16]. Spermatozoa are highly susceptible to cryoinjury; the reactive oxygen species (ROS) released during cooling induce oxidative DNA damage in bovine spermatozoa [17]. Sperm DNA fragmentation leading to a loss of fertility potential has been reported [18]. To date, the effects of IGF-I on buffalo acrosin proteolytic activity and DNA fragmentation have not been reported. The objectives of the current study were to assess the effect of IGF-I on (1) sperm acrosin proteolytic activity, chromatin decondensation, mitochondrial membrane potential, and acrosomal reaction and (2) in vitro fertility. 2. Materials and methods 2.1. Sample collection Frozen semen samples from Surti buffalo (Bubalus Bubalis) bulls aged between 2 and 6 yr (n = 8, six ejaculates per animal) were used for the current study. All the samples were collected within a month. The bulls were consistently giving ejaculates with a concentration of > 500 million cells/mL, prefreeze motility of >60%, and total abnormality of < 20%. The postthaw motility of the semen was also consistently >40%. The animals were reported to be having good fertility after AI. Two frozen semen straws (each straw containing 20 million

cells) of the same batch from each bull were thawed for 1 min at 37 8C and washed twice to remove IGF-I by centrifugation at 500  g in Tris buffer (0.25 M Tris, 80 mM citric acid, 69 mM fructose, and antibiotics). The IGF-I–washed samples were pooled, mixed well, and divided into two equal parts (one control and another IGF-I). In the IGF-I group, insulin-like growth factor I (rhIGF-I analogue) was added to get a final concentration of 100 ng/mL semen, and the semen was incubated at 37 8C for a period of 2 h. The dose rate of IGF-I was chosen as per the earlier study in this laboratory [4]. The sperm functional parameters were assessed at 30-min intervals during the incubation period. For sperm functional tests, a minimum of 200 cells were counted for each straw. Unless and otherwise stated, all the chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Assessment of acrosin proteolytic activity Gelatin substrate film assay [19] with slight modifications was used to assess the acrosin proteolytic activity (APA) of the sperm. Gelatin-covered slides were prepared by spreading 80 mL 10% gelatin (Himedia, Mumbai, India) in distilled water on the slides, and the slides were air-dried and stored horizontally at 4 8C overnight. The slides were fixed in 0.05% glutaraldehyde in phosphate-buffered saline (PBS; 34.22 mM NaCl, 20.8 mM Na2HPO42H20, 1.42 mM KH2PO4, pH 7.8) containing 15.72 mM a-D-glucose (anhydrous) for 2 min. The slides were washed twice in PBS by dipping the slides for 15 sec each and were rinsed in doubledistilled water for 15 sec. The fixed slides were air-dried and stored at 4 8C for up to 2 d. The IGF-I–incubated semen samples were removed at different time intervals and washed three times in 2 mL PBS at 2000 rpm for 5 min each. The semen samples were smeared on gelatincoated slides using pipette tips and dried in a horizontal position at room temperature for about 5 min. The slides were then placed horizontally in a humidified chamber and incubated at 37 8C for 3 h to allow proteolysis of the gelatin film by acrosin. After incubation, the slides were observed by means of a phase-contrast microscope at  400 magnification. A clear halo around the sperm head indicated APA. The percentage of sperm showing a halo was assessed by counting at least 200 spermatozoa. For each semen sample, randomly selected fields were photographed, and the diameter of lysis was calculated in at least 50 cells showing the halo using Image J version 1.3 image analysis software distributed in the public domain by the National Institutes of Health (NIH, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/).

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2.3. Fluorogenic assessment of mitochondrial membrane potential Fluorogenic assessment of mitochondrial membrane potential (MMP) was made as described earlier [20]. In brief, 3 mL 1.53 mM JC-1 dye (5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethyl-benzimidazolyl carbocyanine iodide) in dimethyl sulfoxide (DMSO) were added to a 100-mL semen sample containing 25  106 sperm/mL and incubated at 37 8C for 30 min. After 20 min incubation, 10 mL of Propidium iodide (PI) (0.27 mg/mL in phosphate buffer) was added to counterstain nuclei of spermatozoa. Then, the cells were washed, resuspended in PBS, smeared, and analyzed with an HBO-50 mercury lamp–illuminated, epifluorescence microscope (Nikon Eclipse 50i, Japan) equipped with a carboxy fluorescein diacetate (CFDA) filter set (excitation filter, 510 to 560 nm; barrier filter, 505 nm). A minimum of 200 spermatozoa were observed at  400 magnification. The JC-1 dye undergoes a reversible change in fluorescence emission from green to red as mitochondrial membrane potential increases. JC-1 dye accumulates in mitochondria as J-aggregates and fluoresces red-orange when the membrane potential is relatively high/or fluoresces green as the monomeric form when the mitochondrial membrane potential is relatively low. Thus, the cells with green fluorescence were considered to have the lowest membrane potential, whereas the cells with yellowish to orange fluorescence were considered to have the highest membrane potential [20].

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2.5. Comet assay Single-cell gel electrophoresis (SCGE) was carried out as per the procedures described by Linfor and Meyers [22] and Singh et al. [23]. 2.5.1. Preparation of slides Poly-L-lysine–coated one-end frosted microscopic slides (Riviera, Mumbai, India) were coated with 50 mL 1% normal melting point agarose in PBS and dried at room temperature (20 to 25 8C) overnight. Spermatozoa were washed three times with PBS (Ca2+- and Mg2+free) at 900  g for 10 min. An aliquot of sperm cells was treated with 100 mM H2O2 for 1 h at 4 8C for positive control. Sperm samples were mixed with 75 mL (1  105 cells) 0.5% low-melting-point agarose at room temperature, rapidly pipetted on top of the agarose layer, and covered with a glass coverslip. After solidification, the coverslips were removed gently, and another layer of 100 mL 0.5% low-melting-point agarose was pipetted on top of the second layer then covered with cover glass and allowed to solidify at 4 8C. 2.5.2. Lysis of the cells Lysis of the spermatozoa was carried out at 4 8C by immersing the slides for 3 h in a freshly prepared cold lysis solution (0.3 M NaOH, 2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10, with 1% Triton X-100 and 1.0% sodium lauryl sarcosine added just before use). At 2 h incubation, 5 mM DTT was added into the lysing solution.

2.4. Nuclear chromatin decondensation Nuclear chromatin decondensation (NCD) was carried out as per the procedure described by Rodriguez et al. [21], with slight modification. Suspensions of 0.1 mL semen in 0.4 mL saline (0.95% NaCl) were centrifuged twice at 250  g for 5 min until the supernatant appeared clear. The pellet was then resuspended in 0.1 mL sodium borate buffer (pH 9.0) containing 2 mM dithiothreitol (DTT) and incubated at room temperature (20 to 25 8C) for 45 min. To the incubated suspension, 0.1 mL of freshly prepared 1% sodium dodecyl sulfate (SDS) in sodium borate buffer was added and the pellet resuspended gently then incubated for 2 min at room temperature. The cells were fixed by adding 0.5 mL 2.5% glutaraldehyde in sodium borate buffer to the incubated semen sample. A minimum of 200 cells were counted under high power (400) in a phase-contrast microscope to determine the proportion of cells with decondensed (expanded) nuclei.

2.5.3. Electrophoresis The slides were washed with double-distilled water and kept for 20 min in a horizontal gel electrophoresis tank filled with electrophoresis solution containing 300 mM NaOH and 1 mM EDTA, with pH 13.0 at 12 to 15 8C for 20 min. The electrophoresis buffer level was adjusted at a level of 3 mm above the slide surface, and electrophoresis was performed at 15 8C for 20 min at 12 V. After electrophoresis, the slides were drained, washed, and flooded with neutralization buffer (0.4 M Tris HCl, pH 7.5) for 15 min. Then, the slides were stained with 200 mL ethidium bromide (EtBr) (10 mg/mL dissolved in distilled water) for 10 min. In the randomly selected fields, the individual cells or comets were viewed using a Nikon epifluorescence microscope (400 magnification), and digital comet images were captured. The comet tail length and the DNA fragmentation (%) of at least 50 cells in each semen sample were determined using comet score software (freeware). The percentage of DNA in the head

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is defined as (S pixel intensities in the head/S total pixel intensities of the entire comet)  100. The percentage of DNA in the tail is defined as (S pixel intensities in the tail/S total pixel intensities of the entire comet)  100. Tail moment is defined as the product of the amount of DNA in the tail with the distance between the means of the head and tail distributions. Olive tail moment is calculated as the summation of tail intensity profile values (tail intensity profile equals the vertical summation of the pixel intensities in the comet tail) by their relative distances to the center of the comet head, divided by total comet intensity. The olive tail moment has been used to quantify DNA damage. 2.6. Acrosomal reaction The acrosomal reaction was assessed as per the procedure described by Giritharan et al. [24]. The effect of IGF-I on the capacitation was assessed by the ability of the spermatozoa to undergo acrosome reaction.Two 0.25mL straws of each frozen semen sample were thawed in a water bath at 37 8C. Capacitation was carried out by first washing (225  g for 5 min) the spermatozoa in Brackett and Oliphant (BO) medium supplemented with 0.6% bovine serum albumin (BSA) and 10 mM caffeine– sodium benzoate followed by incubation (38.5 8C, 5% CO2, 95% relative humidity) in the capacitation medium for a period of 4 h. The percentage of capacitated sperm was observed at 2 and 4 h by fluorescein isothiocyanateconjugated (FITC) Pisum sativum agglutinin (FITCPSA), and the spermatozoa were counterstained with PI. At the end of the incubation, the samples were mixed thoroughly, smeared on a grease-free slide, and fixed with methanol for 10 min. The slides were washed three times with DPBS (Dulbecco’s modified phosphate-buffered saline) media containing 0.6% BSA. Fifty microliters of freshly prepared staining solution containing fluorescein isothiocyanate-conjugated (FITC) Pisum sativum agglutinin (FITC-PSA) (5 mg/mL) and PI (7 mg/mL) was poured on the slides and incubated in the dark for 25 min. Then the slides were washed three times with DPBS and dried in the dark room. A drop of 80% glycerol in DPBS was kept on the stained area, covered with coverslip, and sealed tightly with polish. At least 200 cells were counted for each sample under fluorescent microscope (Nikon Eclipse E50i) with excitation filter of 510 to 560 nm and barrier filter of 505 nm. The cells that did not have a clear acrosomal cap were considered as acrosomal reacted. The percentage of acrosomal-reacted sperm cells due to treatment was calculated by subtracting the percentage of acrosomal-reacted cells at 0 h from the percentage of acrosomal-reacted cells at 4 h.

2.7. In vitro fertility 2.7.1. Cleavage rate The assessment of cleavage rate was carried out as per the procedure described earlier [20]. In vitro fertilization was performed in four replicates. In total, approximately 100 (25 in each replicate) matured buffalo oocytes with an evenly granulated cytoplasm and expanded cumulus were used for assessing the effect of IGF-I on fertility. The source of the oocytes in this study were the buffalo ovaries collected from a local abattoir and transported to the laboratory within 2 h in warm (32 to 35 8C) normal saline supplemented with 50 mg/mL gentamicin. Cumulus-oocyte complexes (COCs) from these ovaries were collected by aspiration from nonatretic follicles of 3- to 5-mm diameter using an 18-gauge needle and 10-mL syringe into aspiration medium comprising 5 mL TCM-199 with an equal amount of DPBS containing 0.3% BSA and 50 mg/mL gentamicin. The COCs with 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation. The selected oocytes were washed once with the aspiration medium and twice in the in vitro maturation medium consisting of TCM-199 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 0.2 mM sodium pyruvate, 0.01 U/mL follicle-stimulating hormone (FSH; Armour FSH standard G-94, Sigma, Bangalore) from porcine pituitary, 0.012 U/mL luteinizing hormone (LH; NIH standard LH-525, Sigma, Bangalore) from ovine pituitary, and 1 mg/mL estradiol17b and 50 mg/mL gentamicin. Oocytes in groups (8 to 12) were transferred into 50-mL droplets of maturation medium in Petri dishes, covered with warm (38.5 8C) mineral oil, and placed in a CO2 incubator (38.5 8C, 5% CO2 in air and 90% to 95% relative humidity) for 24 h. After 24 h incubation, the oocytes were examined for their cumulus expansion, and the fully expanded COCs were used for in vitro fertilization. The COCs were prepared for fertilization by washing them in BO medium supplemented with 0.5% fatty acids–free BSA, 2.5 mM caffeine–sodium benzoate, and 10 mg/mL heparin. The randomly selected semen straws from three different bulls were thawed at 37 8C, pooled, and washed twice by centrifugation at 500  g for 5 min in BO medium (without BSA) containing 10 mg/mL heparin. The pellets were resuspended in 0.5 mL BO medium for fertilization. In the IGF-I group only, IGF-I was added to a final concentration of 100 mg/mL of semen in BO medium for fertilization. The samples were placed in CO2 incubator (38.5 8C) for 120 min. At the end of sperm swim-up, the top 400 mL from each tube

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Table 1 Effect of physiologic concentration of IGF-I (100 mg/mL) on acrosin proteolytic activity, mitochondrial membrane potential, and nuclear chromatin decondensation in spermatozoa (mean  SE) incubated in vitro for a period of 2 h. Duration (min)

0 30 60 90 120

Acrosin proteolytic activity (%)

Mitochondrial membrane potential (%)

Nuclear chromatin decondensation (%)

Control

IGF-I

Control

IGF-I

Control

IGF-I

30.85  1.08 22.71  0.57a 20.33  0.91 16.76  1.18 16.26  1.02

29.70  1.02 26.13  1.03b 23.36  1.30 17.54  0.99 16.54  1.05

43.78  1.48 26.71  1.02a 18.77  2.09 16.92  1.24a 11.77  1.30

45.30  1.70 33.27  2.62b 24.24  3.45 22.86  3.02b 17.23  3.02

66.98  3.61 75.50  2.74 80.01  2.43 82.57  2.58a 85.88  2.19a

66.88  3.61 75.80  1.89 76.76  1.19 80.54  2.36b 81.31  3.24b

a,b

Values with different superscript letters between groups for a particular attribute differ significantly.

was aspirated, and the similar groups of samples were combined. The combined samples were centrifuged (500  g) for 5 min. Supernatant was discarded leaving 500 mL at the bottom, and the concentration was adjusted to 5  106 sperm/mL with BO medium for fertilization. The matured oocytes were transferred into 100 mL of semen droplets, overlaid with mineral oil, and incubated for 16 to 18 h. After incubation, the BO medium and unattached sperm were removed and replaced by TCM199 supplemented with 10% steer serum along with 10 to 15 motile buffalo oviductal cells and further incubated for 24 h. After 40 to 42 h of insemination, presumptive zygotes were evaluated under stereo zoom microscope for evidence of cleavage. The cleavage rate was determined by dividing the number of oocytes cleaved out of the total number of oocytes inseminated. 2.7.2. Sperm–zona pellucida binding The sperm–zona pellucida binding was performed in four replicates as described earlier [20] with slight modification using immature good and very good quality COCs. In total, 100 immature COCs were used for each bull. The semen droplets (100 mL) were prepared in a similar way to that for in vitro fertilization, and oocytes (8 to 10 oocytes/drop) were added and covered with mineral oil. The gametes were incubated at 38.5 8C in 5% CO2 in air and 90% to 95% relative humidity for 18 h. After incubation, the oocyte-sperm complexes were rinsed and vortexed for 3 min in DPBS containing 0.5% BSA to remove loosely attached sperm. Then the oocyte-sperm complexes were fixed in 2.5% glutaraldehyde in DPBS and stained with 10 mL PI (0.27 mg/mL in DPBS) in 100 mL DPBS containing 0.5% BSA for 10 min each. The sperm-oocyte complexes were mounted on slides, slightly compressed under coverslip, supported with Vaseline (petroleum jelly, Himedia, Bangalore) at the four corners, and sealed. The prepared slides were kept in a humidified chamber protected from light until examined under epifluorescence microscope (Nikon Eclipse 50i). The number of sperm bound to an oocyte was counted.

2.8. Statistical analysis The percentage data were arcsine square-root transformed before analysis. Statistical analysis was performed by using SPSS software (SPSS Inc., Chicago, IL, USA). Data of all the six ejaculates were pooled for each animal, and the mean values were used for statistical analysis. Statistical significance between groups was determined using Student’s t-test, and within-group significance was determined by one-way ANOVA. All the values are expressed as mean  SE. Statistical significance was accepted at P < 0.05.

Fig. 1. Effect of physiologic concentration of IGF-I (100 mg/mL) on acrosin proteolytic activity (APA) of buffalo spermatozoa incubated in vitro for a period of 2 h. (A) Halo formation indicating APA by gelatinolysis (arrow with dashed line points to more APA, and arrow with solid line points to no APA). (B) The sperm acrosin proteolytic activity has been assessed by measuring the halo diameter (mM) (*P < 0.05).

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3. Results 3.1. Acrosin proteolytic activity Treatment with IGF-I had no effect on acrosin proteolytic activity compared with that of the control (Table 1). However, the acrosin activity (the mean diameter of halo formation in micrometers; Fig. 1A) was significantly higher in the IGF-I group (14.17  1.51) compared with that of the control (9.50  0.36) at 2 h incubation (Fig. 1B). 3.2. Mitochondrial membrane potential

Fig. 2. Effect of physiologic concentration of IGF-I (100 mg/mL) on mitochondrial membrane potential of buffalo spermatozoa incubated in vitro as assessed by JC-1 staining. The membrane potential differs significantly at 30, 60, and 90 min incubation.

The mitochondrial membrane potential in the IGF-I group was significantly higher than that in the control group at 30 min (33.27  2.62% vs. 26.71  1.02%), 60 min (24.24  3.45% vs. 18.77  2.09%), and 90 min (22.86  3.02% vs. 16.92  1.24%) incubation (Fig. 2). However, at 2 h incubation, no significant difference was observed between the IGF-I (17.23  3.02%) and the control (11.77  1.30%) groups.

groups at 2 h compared with that at 0 min incubation. However, it did not differ significantly between the IGF-I and the control groups at 2 h incubation. The tail and olive tail moments were significantly higher in the control at 2 h compared with those at 0 min incubation. However, the tail and olive tail moments in the IGF-I group did not differ significantly from those in the control group at 0 and 2 h incubation.

3.3. Sperm nuclear chromatin decondensation The percentage of spermatozoa positive for NCD did not differ significantly between the groups up to 60 min incubation, but it differed significantly between the groups at 90 and 120 min incubation (Table 1).

3.5. Acrosomal reaction The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 2 h (19.36  4.61% vs. 17.89  4.28%) and 4 h (41.12  6.44% vs. 43.53  5.05%) incubation (Fig. 4).

3.4. Comet assay The effect of IGF-I on comet parameters of buffalo spermatozoa are presented in Table 2. In the control, the comet length increased significantly at 2 h incubation compared with that at 0 min incubation. Further, at 2 h incubation, the comet length was also significantly greater in the control compared with that of the IGF-I group. The percentage of fragmented DNA (Fig. 3) in the tail was significantly higher in the control and the IGF-I

3.6. In vitro fertility The number of spermatozoa binding to the oocyte did not differ significantly between groups, though average number of sperms bound to the oocyte was nonsignificantly higher in the IGF-I group (5.50  1.36)

Table 2 Effect of physiologic concentration of IGF-I (100 mg/mL) on comet parameters (mean  SD) of buffalo spermatozoa incubated in vitro for a period of 2 h. Comet parameters

0 min

2h Control

Comet length (mm) Intact DNA (%) (Head DNA %) Fragmented DNA (%) (Tail DNA %) Tail moment Olive tail moment a,b

a

48.135  4.45 83.56  1.75a 16.44  1.75a 4.34  0.58a 4.42  0.59a

Values with different superscript letters within a row differ significantly.

IGF-I b

72.66  7.15 68.77  2.32b 31.23  2.32b 19.62  5.35b 16.29  3.68b

53.60  4.51a 69.17  3.97b 30.84  3.97b 16.54  4.80a,b 12.72  3.19a,b

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Fig. 3. Effect of physiologic concentration of IGF-I (100 mg/mL) on DNA fragmentation of buffalo spermatozoa incubated in vitro for a period of 2 h by using alkaline comet assay. Tail formation indicates DNA fragmentation.

Fig. 4. Effect of physiologic concentration of IGF-I (100 mg/mL) on acrosomal reaction of buffalo spermatozoa. The percentage of acrosomal-reacted sperm was observed at 2 and 4 h by FITC-PSA and counterstain with PI.

than that in the control group (4.84  0.92). The cleavage rate was significantly higher in the IGF-I– treated group (56.73  3.70) than that in the control group (44.85  2.15). 4. Discussion Fertility is equally related to the quality of the spermatozoa and oocytes apart from appropriate conditions for fertilization and embryo development. In recent years, few studies have been focused on improving the quality of bovine spermatozoa by addition of IGF-I in vitro [3,4]. The current study has been focused to assess the effect of IGF-I on improving spermatozoa functional parameters involved in the fertilization process.

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Maintaining the molecular basis of male fertility is essential for sperm-egg interaction and embryo development. The quality of the acrosomal enzymes has to be preserved during the process of semen preservation and transport in the female reproductive tract until fertilization. Acrosin activity has been measured by the gelatinolysis technique, which is a more effective method for evaluating acrosomal membrane damage [25]. APA has been positively associated with zona pellucida binding and penetration [26,27]. Moreover, acrosin has been involved in capacitation, acrosome reaction, and chromatin decondensation during male pronucleus formation [28,29]. The absence or reduced activity of acrosin has been reported in human spermatozoa with unexplained infertility [15,30]. In the current study, a positive effect of IGF-I on acrosin proteolytic activity suggests that IGF-I maintains acrosomal membrane quality. Mitochondrial membrane potential is widely used for characterization of cellular metabolism, viability, and apoptosis in various cells. A positive correlation exists between the sperm mitochondrial function, motility, and fertility [20,31–35]. In the current study, addition of IGF-I prevented deterioration of sperm mitochondrial membrane potential. Motility of spermatozoa depends on the integrity of the mitochondrial sheath, of which phospholipids are a major component. If fatty acids in these phospholipids are oxidized by free oxygen radicals, spermatozoa will be damaged [36] and their motility will be impaired [37]. Insulin-like growth factors in the neuronal cells prevent mitochondrial dysfunction, maintain calcium homeostasis, and increase cell survival [38,39]. The reduction in mitochondrial membrane potential defines an early stage of apoptosis preceding other manifestations of this process such as DNA fragmentation, ROS production, and the late increase in membrane permeability [40]. Because determination of mitochondrial membrane potential reflects cellular metabolism, viability, and apoptosis, the protective role of IGF-I on mitochondrial membrane potential in the current study clearly suggests the beneficial role of IGF-I on spermatozoa function. Chromatin condensation and stability are the critical factors influencing fertility while using frozen semen [41]. The percentage of cells positive for NCD differed significantly between groups at 90 and 120 min incubation. Condensation of the chromatin during spermatogenesis and epididymal transport and its decondensation at the time of fertilization are essential for successful fertilization. In ram, fertility has been positively correlated with stability of sperm chromatin

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in vitro [21]. The addition of IGF-I decreased the sperm nuclear decondensation in the later part of incubation, but improved the cleavage rate suggesting that the study of NCD in relation to fertility remains controversial. The mechanisms involved in the changes in chromatin stability in spermatozoa are not clearly understood. Jager et al. [42] observed that decondensation depended on time, temperature, and pH, suggesting that human sperm decondensation is more a physicochemical process. Sperm zinc protects an inherent capacity for decondensation due to zinc-thiol interaction in the chromatin [43]. However, the role of IGF-I in reducing chromatin decondensation remains to be established. Integrity of the sperm DNA is the more vulnerable element of sperm cell biology. Hence in the current study, SCGE was used for detecting primary DNA damage at the individual cell level to study the effect of IGF-I on spermatozoa DNA quality. Sperm DNA integrity is linked to male infertility. To our knowledge, information on the comet characteristics of buffalo bull semen has not been reported for fresh semen or for that after freezing/thawing. In the postthaw buffalo semen samples, we observed about 16.44% DNA fragmentation. This is similar to the findings reported in cattle [44]. However, this is in contrast with boar [45] and equine [46] spermatozoa where much higher levels of DNA fragmentation are reported. Low levels of DNA fragmentation after cryopreservation are a characteristic feature of bull spermatozoa and can be a part of the remarkable cryoresistance of bull spermatozoa. It has been reported that the buffalo spermatozoa are more susceptible to cryoinjury. This study suggests that the cryoinjury in the buffalo spermatozoa may affect only the membrane integrity rather than DNA fragmentation. DNA damage is particularly important as it correlates not only with impaired conception rates but also with the health and well-being of the offspring [47]. In the current study, the lower DNA fragmentation observed after IGF-I treatment suggests the protective effect of IGF-I on DNA fragmentation. Earlier studies report that IGF-I acts as an antioxidant and is capable of maintaining functional membrane integrity in vitro [4]. It has also been established that ROS generation and redox balance are the most important factors responsible for disruption of condensation and stability of the sperm chromatin after cryopreservation. Though the sperm DNA is damaged at low levels of oxidative stress, the spermatozoa exhibit a normal or slightly enhanced capacity for fertilization. Oocytes and embryo can repair DNA damage, but there is a threshold beyond which the sperm DNA cannot be repaired [48] to maintain embryo quality [49]. However, at high levels

of oxidative stress, infertility may result due to collateral peroxidative damage to the sperm plasma membrane, compromising the capacity for movement and/or sperm-oocyte fusion. Initiation of sperm capacitation is related to an alteration in the redox balance between ROS generation and the activity of the antioxidant defense mechanisms [1]. Sperm binding to the zona pellucida and the cleavage rate have been used to assess semen function [50]. In the current study, the buffalo immature oocytes with cumulus cells (very good and good quality grade) were used for sperm-zona binding test. Though the average number of sperm binding per oocyte was higher in the IGF-I–treated group, no significant difference was observed between the groups. However, significantly higher cleavage rate in the IGF-I–treated group suggested a positive role of IGF-I on fertility. Failure of sperm-zona binding is mainly due to abnormal sperm functional parameters rather than the oocyte quality [51]. For successful fertilization and embryo survival, apart from oocyte factors, the specific spermatozoa functions must be fulfilled optimally [52,53]. It is established that the functional parameters of spermatozoa are more or less independent of each other [11,54,55]. If even one parameter is suboptimal, fertilization might be affected. In the current study, we have observed that incubation of spermatozoa with IGF-I before insemination improved cleavage rate. A better cleavage rate may be due to the stability in mitochondrial membrane potential and spermatozoa DNA quality during incubation. The potential activity of IGF-I is controlled mainly through expression levels of receptors and IGF-I binding proteins, as the bioavailability and effects of IGFs are modulated by IGF binding proteins (IGFBPs) through competition with IGF receptors for IGFs. Hence, the role of IGF-I on semen quality and fertility have to be considered in relation to IGFBPs [56]. The role of IGFBPs in buffalo remains to be characterized. To understand these interactions, all components of the IGF system have to be included in future studies on the role of IGFs in fertility of bulls. This study provides evidence that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility. This study also suggests that the addition of IGF-I to the extender before freezing may improve fertility in subfertile semen. Acknowledgments The authors thank the Director, National Institute of Animal Nutrition and Physiology (NIANP), ICAR

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(Bangalore, India), for providing the necessary facilities to carry out this work. The authors also thank the project trainees of NIANP for extending technical help to carry out this work.

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