Seminal Plasma Osteopontin: A Marker for Potential Fertility in Dogs

Physiological similarities of dogs with wild canines and humans feature them as an ideal experimental model for humans. Conventional semen parameters such as concentration, volume, motility, live and dead spermatozoa and sperm morphology in the whole ejaculate are frequently examined to assess its quality. There are limited number of studies pertaining the interrelationship between fresh semen characteristics and fertility in canines. Several investigations reveal that mean seminal parameters are poor indices of fertility in dogs (Oettle, 1993). Different isoforms of OPN were demonstrated in the SP and sperm membrane of dogs with unknown function (Souza et al., 2009). Kumar et al., (2017) documented the levels of seminal OPN, Total Antioxidant Capacity (TAC) and MDA in high-fertile and subfertile buffalo bulls. However, there are no reports currently available regarding the endogenous levels of OPN in the SP of dogs that relate to fertility. Therefore, the prime objective of this study was to evaluate the sperm microscopic characteristics in dogs brought for breeding soundness examination and to quantify the endogenous OPN concentration in the SP of dogs and to study its relationship with the sperm in vitro characteristics.

The study was conducted during August, 2018 to July, 2019 on 21 male dogs of different age groups and breeds which were presented for semen quality evaluation at Madras Veterinary College Teaching Hospital, TANUVAS, Chennai, Tamil Nadu, India. 8 dogs displayed conception failure within atleast 3 mating’s with different bitches of similar age and weight and were classified as hypofertile and the remaining 13 dogs had normal conception rates on mating with different bitches and were categorized as fertile. Semen was collected by digital manipulation technique. The pre-sperm and post-sperm fractions were discarded while the sperm rich second fraction was kept in water bath at 37°C for further evaluation. The samples were divided into two aliquot, one for estimation of microscopic characteristics and another for harvesting the SP for quantifying the levels of OPN, MDA and different antioxidant enzymes. Fresh ejaculates were immediately analyzed for progressive motility, viability, morphology, plasma membrane integrity and acrosomal integrity as per Kidd et al., (2001). DNA integrity status (Fig 1) was evaluated by fluorescent staining by Acridine Orange (AO) dye (concentration - 0.19 mg/mL; TC262 Himedia Labs). JC-1 cationic dye (T4069, Sigma Aldrich) was used for assessment of the sperm mitochondrial membrane potential (MMP) under fluorescent microscope (Fig 2). The OPN concentration in the canine SP was estimated by canine OPN ELISA kit (Catalogue No: E1781031 (Type II), Sincere Biotech, Beijing, China) as described by the manufacturer. The concentration of MDA and antioxidant enzymes namely CAT, GPx and SOD were also quantified by canine specific ELISA kits (Sincere Biotech, Beijing, China).
 

 

       
Statistical package for social sciences programme version 23 (IBM SPSS) was used for statistical analysis. Numerical data were expressed as mean±S.E. Data were divided according to fertility status of the dogs. When comparing the fertile group with hypofertile group, the statistical significant differences among the different parameters were obtained using the independent Student’s t test. Pearson’s correlation was used to study the relationship between different variables. P<0.01 and P< 0.05 were considered highly significant and significant at 99% and 95% confidence interval respectively. Linear regression model was used to identify the degree of dependency of OPN on evaluated sperm characteristics.

Sperm microscopic characteristics comparison between fertile and hypofertile group
 
Significantly lower sperm motility in Group II (58.12 ± 6.31%) may be either due to primary or secondary abnormalities of the male accessory sex glands or primary abnormalities that existed in the sperm (Table 1). Significant increase in the percentage of viable spermatozoa in Group I (77.45 ± 2.56%) when compared to Group II (51.68 ± 7.55%) was within the reference values as per Robert et al., (2016). Higher percentages of morphologically normal spermatozoa in fertile dogs (73.72 ± 2.16%) comply with the findings of Oettle (1993). Significantly higher percentage of plasma membrane intact spermatozoa in Group I (78.62 ± 1.88%) as against Group II (57.06 ± 6.15%) were slightly higher than the values reported by Majumdar et al., (2001) in their study in human subjects. Higher acrosomal intact spermatozoa in Group I (72.32 ± 2.01%) when compared to Group II (54.74 ± 5.74%) coincides with the reports of Emokpae and Uadia (2006), who reported a strong relationship between fertilizing ability and acrosomal enzyme activity of the spermatozoa. Significant difference in the DNA integrity status between Group I (81.56 ± 2.30%) and Group II (67.11 ± 7.14%) are in agreement with the findings of Evenson et al., (1980). Higher levels of sperm DNA damage in hypofertile dogs indicate that reduced fertility in male dogs might be associated with ejaculates having poor DNA integrity. Decrease in sperm MMP of Group II (66.47 ± 8.65%) when compared against Group I (86.98 ± 1.48%) could be associated with increased production of ROS. Furthermore, MMP status may be altered during early stages of apoptosis. Therefore, the interrelationship could be due to ROS generation from damaged mitochondria, in turn damaging the mitochondrial membrane (Quillet-Mary et al., 1997).
 

 
Endogenous OPN concentration
 
Current study depicted a significant (P<0.05) increase in the OPN concentration (ng/mL) in Group I (16.36 ± 0.47) when compared to Group II (13.57 ± 0.92). This study estimated the endogenous OPN level in the SP of dogs for the first time by ELISA method. Similar to the present study, Waheed et al., (2013) and Waheed et al., (2015) documented the concentration of OPN in the SP of stallion and camel respectively and found it significantly higher in the high-fertile group as compared to low-fertile group. It was also reported that OPN contains two heparin binding domains and a calcium binding site. Due to the presence of heparin binding domain, heparin from the oviductal epithelium attaches to the OPN molecule. The complex thus formed was responsible for guiding calcium transients which played a pivotal role in the process of sperm capacitation (Boccia et al., 2013). So, it can be suggested that fertile dogs had higher number of OPN receptors available on the sperm surface for its attachment to the heparin binding domain, in turn resulting in increased fertility potential. By these findings we can arrive at a conclusion that identification of SP proteins such as OPN can be potentially used as a fertility indicator in dogs although further studies on different age groups and breed with a greater sample size is needed to substantiate current findings.
 
MDA concentration
 
Significant increase in MDA concentration (ng/mL) in Group II (69.18 ± 1.95) when compared against Group I (63.39 ± 0.85) were consistent with the findings of Colagar et al., (2009). Such findings confer that higher levels of MDA in the SP of hypofertile dogs was indicative of oxidative stress which can be associated with decrease in sperm quality parameters, thus lowered fertility rates.
 
Antioxidant enzymes
 
Contents in several literatures are still debatable as to whether defective antioxidant defense in the SP contribute in generating high semen ROS levels in some infertile subjects. The results from this study (Table 1) are keeping with the reported observation by Zini et al., (1993). On the other hand, current findings are in conflict with a number of reports suggesting that defective seminal antioxidant capacity was indicative of oxidative stress (Lewis et al., 1995 and Alkan et al., 1997).
 
Relationship of endogenous OPN level with sperm characteristics (table2)
 

 
Current study revealed a highly significant positive correlation between the endogenous OPN level and sperm motility in the pooled samples and are in association with the reports of Moura et al., (2007). Highly significant positive correlation between OPN level and the percentage of viable spermatozoa in the pooled samples might be due to the fact that OPN has been described as a cell survivability factor that protected cells from apoptosis. This anti-apoptotic property and enhancing cell survivability was through the activation of integrins and CD44 membrane receptors and signal transduction mechanisms, including the activation of different pathways especially Map kinase and phosphoinositide (PI) 3- kinase (Lee et al., 2007).
       
Highly significant positive correlation between OPN level and the percentage of morphologically normal spermatozoa are in keeping with the reports of El Haggar et al., (2013). Erikson (2006) conducted in vitro studies but could not establish any direct relationship between the OPN concentration and morphological aspects of sperms. Significant positive correlation between OPN level and the percentage of plasma membrane intact spermatozoa were in accordance with the findings of Baruah et al., (2017) that documented the role of OPN in stabilizing the sperm plasma membrane and resist damages occurring during cryopreservation process. Highly significant positive correlation between OPN level and the percentage of acrosomal intact spermatozoa simulate the findings of Cancel et al., (1999) who stated that the amount of OPN was more in acrosomal intact spermatozoa and vice versa. There were no previous reports to substantiate highly significant positive relationship between the endogenous OPN level and the DNA integrity status of the sperm cells in pooled samples. Erikson (2006) in an in vitro study suggested that in the presence of OPN there was an increase in the MMP status of the spermatozoa which might explain the positive correlation between the endogenous OPN level and sperm MMP.
 
Regression of OPN on microscopic sperm characteristics (Table 3)
 

 
Linear regression analysis depicted a highly significant influence of  OPN concentration (ng/mL)  on percent sperm motility (β = 0.860, t = 7.335, P<0.001); viability (β = 0.742, t = 4.826, P<0.001); sperm morphology (β = 0.782, t = 5.464, P<0.001); plasma membrane integrity (β = 0.853, t = 7.127, P<0.001); acrosome integrity (β = 0.866, t = 7.564, P<0.001), DNA integrity (β = 0.783, t = 5.494, P<0.001) and sperm MMP (β = 0.903, t = 9.177, P<0.001).

The comparison of different parameters of canine spermatozoa in dogs with different fertility potential has been elucidated in this study. Decreased seminal attributes in dogs with reduced fertility in comparison with normal fertile dogs will technically assist in breeding where the fertility data cannot be generated for the female counterpart. The present study is first of its kind to document the endogenous levels of seminal OPN and its relation to fertility in dogs. OPN detection by ELISA is simple, cheap and economical which can be readily applied in screening dogs for fertility. Particular biomarker pattern should be characterized in the fertile and hypofertile semen samples. Future efforts must be focused on how these seminal biomarker correlate with male infertility issues brought about by interaction of various factors such as heat stress, malnutrition, trauma and diseases.