An Investigative Immunoassay Targeting Two Osteopontin Epitopes in Boar Semen

The identification of superior sire is largely dependent on the semen quality evaluations, currently through computer-aided sperm class analyser (CASA). Immunoassays present a viable method for analysis of proteins associated with fertility, such as osteopontin. We have targeted osteopontin at an upstream epitope at position 249 - 267 amino acids and a downstream epitope at position 59 - 74 amino acids of full osteopontin protein in raw, BTS, Kobidil, Tris and Citrate liquid preserved Large white and Kolbroek boar semen. The Genetool analysis software was used to quantify the amount of osteopontin detected. Three boars per breed were used and experiments were repeated five times. Our results revealed that the downstream 15 amino acid epitope was more sensitive to osteopontin antibody. Between total motility and osteopontin, Kolbroek boar semen preserved in Kobidil, Citrate, BTS and Tris extenders revealed positive correlations of 0.564, 0.471, −0.960 and 0.330, respectively. Large white boars semen showed correlations of −0.043, 0.655, 0.778, and 0.202 Kobidil, Citrate, BTS and Tris, respectively. For rapid motility and osteopontin, Kolbroek boar semen revealed positive correlations of 0.842, 0.601, 0.164 and 0.202, respectively while large white boars semen showed correlations of −0.909, −0.144, −0.210, and 0.089, respectively. Only BTS and Kobidil showed high negative correlations between osteopontin and viability in Kolbroek (−0.975) and Large white (−0.746) boar semen, respectively. No significant correlations with osteopontin were observed when Tris extender was used in both breeds. In conclusion, Kolbroek boar semen requires osteopontin in Kobodil extender while large white boar semen requires osteopontin in BTS extender.


Introduction
The ultimate aim of any pig breeding programme is to increase the profitability of commercial pork production. Artificial insemination practices dominate reproduction management on pig farms in South Africa because it brings superior sire lines into the herd and across many sows [1]. The identification of superior sire is largely dependent on the semen quality evaluations and the reproductive performance of sires [2]. Consequently, boar semen fertility remains an area of interest in the pig farming and industries alike [2] [3]. Large White and Kolbroek boars are popular with our local pig farmers due to their desirable traits including their superior semen motility parameters accompanied by high sperm cell viabilities [4] [5]. The current semen evaluation techniques, such as CASA, provide a very conservative estimate of their relative fertility [4] [5]. These observations have led to interests in the investigations of fertility markers in boars [3] [6] [7] [8]. The seminal plasma proteins, mainly osteopontin, have been recognized as a potential fertility marker in boars [6]. It has been found in the reproductive tract and ejaculated semen, where it modifies semen on route to ejaculation [6] [9]. These sperm modifications by osteopontin influence capacitation, motility and viability of sperm cells [7] [9]. Antibodies against osteopontin revealed that osteopontin was localized in the post-acrosomal region of ejaculates spermatozoa [10] [11] [12]. Also, in adult boars, osteopontin has been found in the testis, specifically, in the early and late germ cells, including spermatids [6] [11]. The most profound illustrated function of osteopontin in boars during fertilization was shown when osteopontin decreased polyspermy, apoptosis and improved fertilization [3] [7]. The structure of osteopontin reveals that it is a long protein of 322 amino acids [13]. It consists of RGD sequence important for adhesion functions and SVVYGG for thrombin cleavage (Figure 1). The down-stream osteopontin epitope at region 59 -74 with 15 amino acids sequence, and the up-stream epitope at position 249 -267 with 18 amino acids sequence of full osteopontin protein, provide great targets for osteopontin immunoassays.
Liquid preservation of boar semen in extenders like Tris, Kobidil, BTS and Citrate, remains a preferred method of storage over short or extended periods [5] [14] [15]. While these extenders have been proven to be ideal for liquid preservation of semen from these breeds, motility parameters are severely compromised over time in an extender and period of incubation dependent manner [4] [5] [16]. Techniques such as ELISA and Western blot analysis can be used to assess the presence or absence of osteopontin in liquid preserved semen [17]. However, in the absence of these techniques, the dot-blot analysis becomes a promising alternative technique. The advantage of using this technique includes the fact that it is simple, quick, and cheap and multiple samples can be analysed at once [17]. The absolute requirement for applying this technique is the use of highly specific antibodies due to the presence of "junk" proteins which might compromise the specificity and sensitivity of the assays hence a densitometric analyser is often required [17]. Genetool software provides a spot blot analysis using a spot grid, and can perform quantitative and incidence analysis plus multiple background noise subtraction.
Tris, Kobidil, BTS and Citrate extenders show low motilities and viabilities over time, is it imperative to investigate whether the osteopontin can be associated with these decreases. Hence, the objective of this study was to determine the correlation between osteopontin detected with total motility, rapid motility and viability of liquid preserved Kolbroek and large white boar semen.

Animal Care
The experiments were carried out at the Pig Research Unit of Germplasm Conservation and Reproductive Biotechnologies Programme at the Agricultural Research Council (ARC), Irene, South Africa. The ARC-AP campus is located at 25˚55' South; 28˚12' East. The campus is located in the Highveld region of South Africa and situated at an altitude of 1525 m above sea level. The experimental protocols were evaluated and approved by the Animal Ethics Committee of the Agricultural Research Council-Animal Production (APIEC13/002).

Semen Collection
Three South African indigenous Kolbroek boars and three exotic Large White boars with ages ranging from nine to ten months were utilized in this study. Boars were in good health and were fed grower diet and water was given ad libitum throughout the duration of the study. Semen was collected once a week for five weeks from each boar by the gloved hand technique, as outlined by [5]. The sperm-rich fraction was collected using a thermos-flask containing warm water (39˚C) and a glass beaker covered with a gauze filter to separate the gel fraction from sperm-rich fraction [4]. Within two hours of collection semen was transported to the laboratory for evaluation. A drop of fresh semen was placed on a microscopic slide and evaluated with the aid CASA system. The semen was diluted using four different short term extenders, namely: BTS, Kobidil, Citrate and Tris-based extenders at a ratio of 1:1 (v/v). The composition of these extenders is shown (Table 1) [4].
Sperm motility and viability were evaluated at 0, 3, 24 and 48 hours (hrs) interval. For viability, cells were stained with SYBR-14/PI as previously demonstrated [18]. Briefly, 50 μl of semen was diluted with pre-warmed BO-Wash to Open Journal of Animal Sciences 1 mL and 5 μl of a 50 times diluted SYBR-14 was added to the cells followed by incubation at 37˚C for 10 minutes. After 10 minutes, 5 μl of propidium iodide was added to the cells followed by incubation for an additional 10 minutes. After 10 minutes, 5 μl of cells were immediately placed on pre-warmed glass slide and observed under a fluorescent microscope (Olympus, model BX51).

Dot Blot Analysis
A dot blot assay is a technique in molecular biology used to detect biomolecules such as proteins using specific antibodies. Freshly collected semen and extended semen was centrifuged at 500 × g for 10 minutes at 5˚C. The precipitated semen was then resuspended in in the semen lysis buffer containing 0.1 M Tris-HCl pH 8.0, 0.14 M NaCl, 10% Glycerol and 1% NP-40. After vortexing, the protein concentration was determined using the Bradford assay and a standard curve was established to determine the semen total protein concentrations [19]. Bovine Serum Albumin (sigma cat#A3311) the standard osteopontin protein (sigma cat#O3514) and the lysates from freshly collected semen was loaded onto the nitrocellulose membrane at protein concentrations of 3 µg/ml, 12 µg/ml and 60 µg/ml at 5 µl per sample. For the liquid preserved semen in BTS, Kobidil, Citrate and Tris-based extenders for 0, 3, 24, and 48 hrs, 5 µl was applied to the high protein affinity nitrocellulose membrane and allow to air dry at room temperature. The high protein affinity nitrocellulose membrane was then blocked for 30 minutes with a 0.1% BSA in 0.05% Tween-20 in 20 mM Tris-HCl, 150 mM NaCl at pH 7.5 (BSA-TBS-T-buffer) solution to prevent non-specific binding of the antibodies to be used in the assay. The membrane was then washed three times for five minutes each with a washing buffer containing 0.05% Tween-20 in 20 mM Tris-HCl, 150 mM NaCl (TBS-T-buffer). The washed membrane was then incubated with a primary antibody, namely, a polyclonal anti-osteopontin antibody raised in rabbits at a dilution of 1:1000 in TBS-T-BSA buffer for 1 hour at room temperature. Two primary antibodies were used, the 15 amino acid antibody corresponding to the 59 -74 amino acid sequence of human osteopontin (Abcam, ab14175) and the 18 amino acid antibody corresponding to the 249 -267 amino acid sequence of human osteopontin (Sigma, O7264). After an hour, the membrane was washed three times for five minutes each with TBS-T. The washed membrane was then incubated with the secondary antibody; the horse reddish peroxidase conjugated anti-rabbit IgG (sigma), at 1:1000 dilutions for 30 minutes at room temperature. The membrane was then washed twice for five minutes each with TBS-T and then incubated with TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) diluted 3,3'-diamino-benzamide-urea-hydrogen peroxide tablets until brown colour development, according to manufacturer's protocol (Sigma, D4168). After colour development, the membrane was rinsed once in TBS and allowed to air dry. The brown colour developed indicated the presence of osteopontin in the semen protein lysates.

Genetool Software Analysis of Osteopontin
The syngene genetool software version 4.03.00 was used to assess the differences

Statistical Analysis
All experiments were repeated 3 times. Pearson's correlation coefficient was used to establish relationships between different parameters. Data were presented as the mean ± SEM. Student's t-test was done wherever applicable, and significance was expressed as a p ≤ 0.05. The syngene genetool software version 4.03.00 was used to assess the differences in the osteoponin levels among the samples. Statistical analyses were done using the software GraphPad InStat Version 3.06 (GraphPad Software).

Results
CASA analysis of boar semen revealed different colours in accordance to semen C. M. Pilane et al. motility properties (Figure 2a). Also, The SYBR-14/PI stain proved definitive in distinguishing between live and dead boar sperm cells (Figure 2b).

Raw semen from Kolbroek and Large White boars show high and comparable
percent total motility and percent viability, however, the rapid motility of Kolbroekboar raw semen was superior to that of large white boars ( Table 2).    Superscripts (a, b) indicates significant difference across the raw between the two osteopontin epitopes.  This indicates that in Large White boar semen, osteopontin is required to maintain motility after 24 hrs in Kobidil liquid preserved semen. On the contrary, in Kolbroek boar semen, osteopontin is not required to maintain motility in BTS liquid preserved semen for the same period. The same is true for Tris liquid preserved Kolbroek semen after 24 hrs. We have established the correlation coefficient in liquid preserved Kolbroek and large white boar semen relating to total motility of osteopontin detected. Our data show that when Kolbroek semen is preserved in BTS, there in high negative correlation between total motility and osteopontin detected (−0.960) while large white boar semen show moderately high positive correlation. In Kolbroek boar semen preserved in Kobidil, Citrate and Tris extenders, positive correlations of 0.564, 0.471 and 0.330, respectively, were obtained. Kobidil preserved semen showed that there was no relationship between total motility and osteopontin detected in large white boars (−0.043) ( Table 5).
Rapid motility of Kolbroek boar raw semen is twice as much compared to large white boar raw semen. Under Kobidil preservation conditions, Kolbroek boars showed a high positive correlation of 0.842 between rapid motility and osteopontin while large white boars showed highly negative correlation of −0.909. When citrate extender is used, Kolbroek boar semen showed positive correlation between rapid motility and osteopontin (0.601) while large white boar semen showed low negative correlation (−0.144) ( Table 6). BTS and Tris revealed low correlation between rapid motility and osteopontin detected in both breeds.
For viability, Kolbroek boars show highly negative correlation (−0.975) between viability and osteopontin when BTS was used while a moderately high negative correlation (−0.746) was observed with Large white boar semen albeit with Kobidil extender (Table 7).  White boar semen showed highly negative correlation with this extender. This observation seems to suggest that osteopontin is required for Kolbroek rapid motility but not in large white boar semen rapid motility.
The citrate extenders show positive correlations between osteopontin and total motility plus viability in both breeds. However, only Kolbroek boar semen shows a positive correlation between Rapid motility and osteopontin. In both breeds, Tris extenders did not reveal any significant correlation coefficient between osteopontin with total motility, rapid motility and viability. Speculation is that for both citrate and Tris, the buffering capacity and calcium chelation when Tris extender is used is compromised hence cell function is impeded.

Conclusion
The role of osteopontin in boars is not understood, however, the fact that osteopontin is present in Kolbroek and Large White boar semen suggests that it might have a role in the boar semen fertilizing ability. Osteopontin is not required for rapid motility in large white boar semen but appears to be required for Kolbroek boar semen rapid motility. This then raises questions about whether the extenders used for semen cryopreservation for these breeds require further scrutiny. The importance of the buffering capacity, chelation capacity and salt content appear to influence motility and viability in a breed dependent manner. This could imply that certain extenders may not be preferable for certain breeds. The limitation of this study is whether these observations can translate to semen fertility when osteopontin is at high levels. To prove this, an in vitro fertilization (IVF) assay must be performed to assess fertilizing ability of boar semen. More research is required to establish the mechanisms of function of osteopontin during boar semen fertilization.