Preservation of Liquid Boar Semen: Effect of Genotype, Boar and Sperm Parameters on Motility and Acrosome Integrity

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1 VETERINARY RESEARCH INTERNATIONAL Journal homepage: ORIGINAL ARTICLE Preservation of Liquid Boar Semen: Effect of Genotype, Boar and Sperm Parameters on Motility and Acrosome Integrity M. H. Khan 1 and Suresh Kumar 2 ICAR Research Complex for NEH Region, Umiam, Meghalaya, India. Present address: 1 Senior Scientist, ICAR-National Research Centre on Mithun, Jharnapani, Medziphema, Nagaland India. Abstract One hundred and forty four ejaculates were collected from 24 mature fertile boars of four different genetic groups (HS, CB-50%, CB- 75% and CB-87.5%). The ejaculates were extended in Beltsville Thawing *Corresponding Author: Solution (BTS). Boar number, boar genotype, volume of the ejaculate and sperm concentration was recorded. Motility and acrosome integrity were M. H. Khan assessed after storage at 18 C for 4, 24, 48, 72, and 120 h. Results revealed that storage time had a significant influence on both motility (p<0.05) and haidermeraj@rediffmail.com acrosome integrity (p<0.001). The average percentage of motility showed a significant decline from 85.6% after 4 h of storage to 72.67% at 120 h. Similarly, the percentage of sperm cells with normal acrosomes declined Received: 02/06/2014 throughout the experiment. The average acrosomal integrity after 4, 24, 48, Revised: 25/06/ , and 120 h of storage were 92.72%, 89.65%, 84.0%, 81.54%, and 75.4%, respectively. The decrease in motility and acrosome integrity from Accepted: 27/06/2015 one storage time to the next was highly significant throughout the trial. There was a significant influence of boar (p<0.005) and sperm concentration (p<0.01) on motility, while acrosome integrity was affected only by boar (p<0.01). There was no influence of genotype or volume of ejaculate on motility and acrosomal integrity of spermatozoa. Keywords: Boar, Genotype, Liquid semen, Sperm concentration, Volume. 1. Introduction Beltsville Thawing Solution (BTS) is one of the most widely used extender for liquid preservation of boar semen. In India, liquid semen is generally being used for AI in pigs. The principal advantage of using unfrozen liquid semen is that the fertility is maintained even with low concentration of spermatozoa in the inseminate. Due to wide variation in fertilizing potential and sperm characteristics of ejaculated semen among the different breeds, fertility rate and conception after AI varies considerably. Apart from genetic effect, there are several other factors which might influence fertility of stored semen like variation among the boar within the same genetic group, composition of ejaculate, volume of seminal plasma and concentration of spermatozoa. Seminal plasma is important for progressive motility of sperm cells. Spermatozoa gain motility during ejaculation as ph and bicarbonate concentration increase during mixing of sperm and seminal plasma (Rath et al., 1989). Further, transfer of sperm cells from seminal plasma to artificial media has shown to decrease motility and increase sperm agglutination (Garner et al., 2001), which indicates that seminal plasma might be of importance to protect membranes and maintain fertilizing capacity during storage. Khan et al. (2010) reported that partial removal of seminal plasma (50-75%) has beneficial effect on motility and viability of spermatozoa during in vitro storage at 20 O C whereas complete removal has some adverse effect on motility and membrane integrity. It has also become evident that seminal plasma is of importance during the process of fertilization. In the female, seminal plasma has a regulatory function on ovulation (Weitze et al., 1990). It has been demonstrated that intrauterine infusion of seminal plasma prior to AI increases fertilization rate (Rath et al., 1989; Watson and Plummer 1985), probably by enhancing passive sperm transport (Rath et al., 1989; Willmen et al., 1989). Sperm concentration also play an important role to maintain motility and acrosome integrity of spermatozoa during storage as It affects the amount of seminal plasma surrounding each spermatozoa in raw as well as extended semen. As sperm concentration increases, the amount of seminal

2 plasma per sperm cell decreases. The aim of the present investigation was to study the influence of breed, sperm concentration and volume of the ejaculate on motility and acrosome integrity of liquid boar semen stored in BTS at 18 o C for 5 days. 2. Materials and Methods 2.1 Semen Collection One hundred and forty four ejaculates from a total of 24 AI boars, aged between 12 and 36 months, were taken for the study. The ejaculates were obtained by including all AI boars scheduled for semen production twice a week, including Hampshire (HS), CB-50% (HS cross with local pigs having 50% exotic inheritance), CB-75% (HS crossed with local pig having 75% exotic inheritance) and CB-87.5% (HS crossed with local pig having 87.5% exotic inheritance). Each group comprised of six boars. All the boars were maintained at Livestock Farm Complex of the institute and were provided with uniform feeding and watering. All the animals were kept as per the guidelines issued by the Institutional Animal Ethics Committee. From each boar, six sperm-rich fractions were collected using the gloved hand method. Semen was filtered through gauze during collection. Sperm concentration was determined by use of a photometer and volume of the ejaculate was recorded with the help of measuring cylinder. Initial extension with BTS (34 C) to approximately one third of the final volume was performed within 15 min after semen collection. The final extension, using BTS (30 C) was performed within one hour after the initial extension. The volume of one insemination dose was 100 ml, and the total number of spermatozoa was estimated to be 2 billion per dose. One AI dose was divided into 5 aliquots and stored at 18 C in plastic vials for further investigation. 2.2 Semen Evaluation Semen samples were re-wormed in a water bath at 35 C for 30 min before examination, which were performed at 4, 24, 48, 72, and 120 hours after semen collection. The examination after 4 h storage was taken as control. Motility was assessed using a phase contrast microscope at 100 magnification and a heating stage (35 C). Six different fields from each sample were examined. The average of all estimates per ejaculate was used for the data analysis. The motility was expressed as percentage of progressively motile spermatozoa. Simultaneously, semen smears from each sample were prepared for determination of acrosomal status using the Geimsa stain (Watson, 1975). After staining, each sample was examined under oil immersion and 1000 magnification using a bright field microscope. From each smear a total of 200 spermatozoa were evaluated for acrosome integrity. The sperm cells were assessed as having normal or altered acrosomes (Oettlé, 1986a,b). The acrosomal integrity was assessed as the percentage of sperm cells with intact acrosome. The spermatozoa showing ruffled or swollen acrosome were not included in the study. 2.3 Statistical Analysis Analysis of variance was applied to determine possible effects of storage time, genotype, boar within genotype, volume of the ejaculate and sperm concentration on semen quality assessments. Data on motility and acrosome integrity assessments were analyzed by the general linear-models. 3. Results and Discussion There is no significant difference in volume of ejaculate as well as sperm concentration between different genetic groups (Table 1). After 4 h of storage, mean percentage of motile spermatozoa was 85.6% which dropped to 67.0% after 120 h in HS boars. The average motility values at 24, 48 and 72 h were 81.56, and 72.50%, respectively. There was no significant difference in motility at 4 and 24 h of storage. However after 24 h, the motility was significantly lower. Similar trend was reported in other genotypes as well. The result of our study is in compliance with earlier investigations (Khan et al., 2006; Waberski et al., 1994). Similar trend were also reported in other genetic groups. Though there was a significant reduction in the motility of spermatozoa during storage, the average motility recorded after 120 h of storage was found to be fairly good and within the acceptable range. Khan et al. (2012) also reported the motility of crossbred boar spermatozoa after 4, 24, 48, 72 and 96 h of storage at 18 o C as 88.25, 75.16, 65.25, and 48.08% respectively preserved in BTS extender which is lower than reported in this study. It might be due to genetic variations between the groups. Similarly, the acrosome integrity, expressed as percentage of sperm cells with normal acrosome morphology, showed a clear decline throughout the experiment. Average acrosomal integrity for 4, 24, 48, 72, and 120 h storage were 92.72, 89.65%, 84.0%, 81.54%, and 75.4%, respectively in HS boar. There was no significant difference between genotypes. There was no significant difference between 4 hours and 24 hours of storage in all the genotypes. However, after 24 hours significant drop in intact acrosomes were observed (Table 2). 31

3 Table 1: Ejaculate volume and sperm concentration in different genetic groups of boars Breed No. of No. of Volume (ml) Concentration boars ejaculates Mean ± SE (million/ml) Mean ± SE HS ± ± CB-50% ± ± CB-75% ± ± CB 87.5% ± ± Table 2: Sperm motility and acrosomal integrity during preservation at 18 o C Sperm parameters (%) (Mean ± SE) Motility Genotype HS Storage (h) ± 3.54 A 81.56±2.78 AB 78.65±3.57 B 72.50± 3.12 B 67.00± 3.19 C CB-50% 83.50±6.51 A 80.25±4.54 A 75.55±6.47 B 70.25±5.65 C 65.45±7.50 C CB-75% 87.54±6.55 A 82.47±7.58 A 73.10±4.98 B 69.45±4.65 B 63.25±4.40 C CB-87.5% 84.15±5.85 A 81.22±3.84 A 74.85±6.48 B 70.50±5.78 B 63.50±6.80 C Acrosome integrity HS CB-50% 92.72± 2.58 A 88.54±3.58 A 89.65±3.12 A 86.25±4.68 A 84.0 ±2.78 B 80.25±6.10 A 81.54±3.16 B 77.56±4.25 B 75.4 ± 3.24 C 73.25±6.50 B CB-75% 89.25±6.54 A 83.70±6.54 AB 79.58±3.54 B 74.50±4.65 BC 72.60±7.54 C CB-87.5% 91.80±4.85 A 87.45±5.40 AB 82.57±5.50 BC 78.50±6.30 C *values with at least one common superscripts (A, B, C, D) did not differ significantly (P<0.05) in a row ±6.53 D Motility is important for semen quality; however, motility alone does not secure fertilizing capacity. Spermatozoa also need intact acrosomes to penetrate the barriers around the ovum. The results from the study indicate that the acrosome is more susceptible to damage during storage than the mid piece of spermatozoa which contain mitochondria responsible for sperm motility. This presumption is in accordance with experiments performed by (Bur, 1990) stating that the decrease of membrane fluidity during storage is greater for head plasma membranes than for sperm body membranes. This is not surprising as storage of diluted semen to some extent may cause sperm capacitation possibly followed by acrosome-reaction (Vishwanath and Shannon, 1997). The decrease in acrosome integrity might thus be due to acrosome reaction in addition to membrane damage (Larsson, 1985). The duration of storage had a highly significant influence on both motility (p<0.01) and acrosome integrity (p<0.001), as was the case for the effect of boar within breed (p<0.005). Sperm concentration affected motility significantly (p<0.01). There was, on the other hand, no effect of either genetic group on motility and acrosome integrity (Table 3). However, genetic group has no influence on either motility or acrosome integrity. These results revealed that there is individual variation among boars concerning preservation of semen quality during storage, and that there seems to be no such variation between the breeds investigated in this study. There might be due to variation in inherent properties of the sperm membranes, possibly being of significance to sperm membrane functionality which might explain the differences between individuals. Effect of semen volume and concentration of spermatozoa per ejaculate was also studied and it was found that there is no influence of volume of the ejaculate on the semen quality parameters investigated during storage. The sperm concentration seems, however, to play an important role for motility but not for acrosome integrity during storage. 32

4 Table 3: Summary of ANOVA for motility and acrosome integrity of sperm cells diluted and stored for 5 days in BTS Source of variation df Level of significance Motility Acrosome integrity Duration of storagege (days) Genotype Boar within genotype Sperm concentration Ejaculate volume The fact that the regression coefficient for sperm concentration in the statistical analysis is negative, demonstrates that the motility is maintained at a higher level during storage when sperm concentration in undiluted semen is low compared to higher sperm concentration. This suggests that there is a positive effect of increasing amount of seminal plasma and furthermore that there might be components in seminal plasma which are beneficial for maintenance of motility, and that the concentration of these components after extension might be important. On the contrary, an adverse effect of seminal plasma was reported on acrosomal integrity of spermatozoa but beneficial effect on motility during in vitro storage at 18 o C (Khan et al., 2010). It was found that when spermatozoa was preserved without dilution at 20 o C for 72 hours had adverse effect of acrosomal integrity but motility was maintained at higher level whereas complete removal of seminal plasma adversely affected the sperm motility but maintained the integrity of plasma membrane. In further studied they reported that 50-75% replacement of seminal plasma with BTS had beneficial effect both on sperm motility and membrane References Bur MM (1990). Preservation of boar sperm alters membrane molecular dynamics. In the Proceeding of the 2nd International Conference on Boar Semen Preservation, Beltsville, Garner DL, Thomas CA, Gravance CG, Marshall CE, DeJarnette JM and Allen CH (2001). Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology, 56: Khan MH, Naskar S and Bardoloi RK (2010). Effect of replacement of seminal plasma on preservation of liquid porcine semen. Indian Veterinary Journal, 87: Khan MH, Naskar S, Das A and Bardoloi RK (2006). Comparative efficacy of different diluents on liquid preservation of boar semen. Indian Journal of Animal Sciences, 76: Khan MH, Nath KC, Deka BC, Naskar S, Bardoloi RK, Bhuyan D and Suresh Kumar (2012). Effect of different extenders on cryo-preservation of Hampshire and integrity. It may be hypothesized that there are some substances in seminal plasma which causes capacitation and acrosome reaction leading to loss of fertility and motility of spermatozoa during in vitro storage becomes less harmful when diluted with BTS or when portion of seminal plasma is removed. 4. Conclusion The results of this investigation revealed that the effect of boar is of great importance concerning semen quality during longtime storage. Further investigation should be done to compare sperm concentration with field fertility data in order, to some extent, to predict individual differences concerning preservation of semen quality during storage of liquid semen. Acknowledgement The facilities provided by the Director of the institute and Head of the Department for carrying out the research work is also highly acknowledged. The technical support provided by the staff of Livestock farm Complex is acknowledged with thanks. crossbred boar semen. Indian Journal of Animal Sciences, 82: Larsson K (1985). Boar sperm viability after freezing and thawing. In the Proceding of the 1 st International Conference on Deep Freezing of Boar Semen, Uppsala, Swedon, Oettlé EE (1986a). Using a new acrosome stain to evaluate sperm morphology. Veterinary Medicine, 81: Oettlé EE (1986b). Changes in acrosome morphology during cooling and freezing of dog semen. Animal Reproduction Science, 12: Rath D, Weitze KF, Pena Alfaro CE and Andrade Moura JC (1989). Effects of seminal plasma on the number of accessory sperm cells and fertilization in gilts. Zuchthyg, 24: Vishwanath R and Shannon P (1997). Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperature. Reproduction Fertility and Development, 9:

5 Waberski D, Meding S, Dirksen G, Weitze KF, Leiding C and Hahn R (1994). Fertility of long-term-stored boar semen: Influence of extender (Androhep and Kiev), storage time and plasma droplets in the semen. Animal Reproduction Science, 36: Watson P F (1975). Use of Giemsa stain to detect changes in the acrosome of frozen ram spermatozoa. Veterinary Record, 97: Watson PF and Plummer JM (1985). The response of boar sperm membranes to cold shock and cooling. In the Proceding of the 1 st International Conference on Deep Freezing of Boar Semen, Uppsala, Swedon, Weitze KF, Rabeler J, Willmen T and Waberski D (1990). Interaction between inseminate, uterine and ovarian function in the sow. Influence of seminal plasma and oestrogens in the inseminate on intragenital sperm transport, time of ovulation and fertility results in gilts. Reproduction in Domestic Animals, 25: Willmen T, Weitze KF, Habeck O and Waberski D (1989). Effect of seminal plasma and different sperm number in the inseminate on fertilization, accessory sperm number and ovulation time. In the Proceeding of the 3rd International Conference on Pig Reproduction, Nottingham, UK. 34