The color of semen was creamy white, while the sperm concentration in Ab1 and Ab2 was, respectively, 958 x 106/ml and 1285 x 106/ml thereby giving an initial impression of normal semen. However, the microscopic semen picture was severely disturbed in Ab1 and Ab2 when evaluated for progressive motility (10 and 15%), viability (35 and 30%), morphological abnormalities (88 and 62%) and HOST (25 and 20%). The predominant morphological abnormalities encountered in Ab1 and Ab2 were mid-piece defects, comprising mainly of Dag defect (32 and 27%), distal mid – piece reflex (DMPR) defect (42 and 17%), and coiled tail (11 and 12%). Sperm viability is directly and significantly correlated to progressive motility and HOST rather than morphology (
Lodhi et al., 2008). Hence, reduced sperm viability justifies a parallel reduction in motility and HOST parameters in the Ab bulls. Dag defect, characterised by strong folding (Fig 1a), coiling and fracture of distal mid piece, is considered to be a Major Sperm Defect as it significantly hampers fertilization potential of the affected sperms (
Chenoweth 2005) both in terms of motility and altered DNA configuration
(Enciso et al., 2011). Semen samples with > 4 percent Dag defect indicates disturbed functioning of testis or epididymis, especially the caudal part and is attributed to faulty genetics or environmental reasons linked to increased dietary Zn (
Barth and Oko, 1989).The DMPR is although considered a Minor Sperm Defect as it has minor effect in impairing fertility. However, a minor defect present in > 15 percent sperms is considered to be a Major Defect, which can be heritable (
Chenoweth, 2005). Further, presence of cytoplasmic droplet within the bend of DMPR (Fig 1b) also confirms a heritable nature of this defect in the Ab bulls. Another possibility of DMPR is contact of semen with hypotonic solution (
Barth and Oko, 1989) which, however, is excluded in present investigation. Based on the microscopic findings the semen samples of the Ab bulls were straightaway rejected and the bulls advised to be culled. However, it was intriguing to investigate the mineral and seminal plasma protein status in the Ab compared to the N bulls.
The status of different minerals in the Ab and N bulls is presented in Table 1. The P content, considered separately in seminal and blood plasma, were similar between the Ab and the N bulls, except for Ab1 having higher numerical value of P (11.5 mg/dl). The P in seminal plasma was higher than the corresponding blood plasma in both Ab and N bulls, which was in line with few previous studies reporting much higher P content in whole ejaculate (477.1 mg/dl;
Gamcik et al., 1992) and seminal plasma (217.5±54.0 stdev) than the blood plasma (18.0 to 32 mg/dl) (
Vrzgula 1990,
Reece, 1998). Higher P in seminal plasma is required as a source of energy for sperms
(Machal et al., 2002). There was no variation in the Ca content of seminal and blood plasma in the Ab
versus N bulls. Alike P, the Ca content in seminal plasma was higher than blood plasma. Compared to present study, much higher Ca content of 153.4 mg/dl in whole semen (
Gamcik et al.,1992) and 63.4±7.8 (stdev)mg/dl in blood plasma (
Machal et al.,2002) has been reported. The Ca in semen has a positive correlation with sperm motility
(Wong et al., 2001) and regulates its capacitation and hyperactivation (
Marquez and Suarez et al., 2004). It is therefore inferred that both P and Ca were not the limiting factors affecting semen quality in the Ab bulls. Na is an important element for sperm functioning
(Mosaferi et al., 2005). Undetectable Na in the seminal plasma of Ab and N bulls could be attributed to individual variation in bulls or ejaculates or its estimation in consecutive ejaculates whereby the Na content increases
(Cragle et al., 1958). It is pertinent to mention here that the ejaculate collection in present study was bi-weekly. Variation in the methodological sensitivities in Na estimation can also not be excluded. However, Na content in blood of the Ab and N bulls is comparable to an earlier study
(Cozzi et al., 2011) and is lower than 181.8 to 238.6mEq/L in cow bulls
(Cragle et al., 1958). K is more concentrated within the sperm cells of bovine semen than seminal plasma. Accordingly, numerically higher K in seminal plasma in the Ab than the N bulls can be attributed to breach in the plasma membrane integrity and thereby leaking of K in the seminal plasma of Ab bulls. K is considered as a natural metabolic inhibitor, its elevation in seminal plasma will reduce sperm metabolism
(Joseph et al., 2013). K values recorded in seminal plasma of present study were lower than a range of 185.1 to 238.6 mEq/L K in normal bulls
(Cragle et al., 1958). However, K in blood plasma in the Ab and N bulls resembled closely to 4.0 mEq/L
(Cozzi et al., 2011). The Cu content in the seminal and blood plasma of the Ab bulls was numerically less that the N bulls. Being a trace mineral, a minute fluctuation in Cu significantly affects its role in bull fertility
(Hedaoo et al., 2008) that includes its positive impact on the progressive motility. Accordingly, Cu was lower in ejaculates with reduced or zero sperm motility in cattle
(Wong et al., 2001) and buffalo
(Dhami et al., 1994) bulls. Low Cu in seminal plasma could be one reason for reduced values of progressive motility in the Ab compared to N bulls. The Zn concentration in seminal plasma of Ab and N were much higher than blood plasma, which corroborates to a negative correlation between semen and blood
(Dhami et al., 2001). Zn is required for membrane and chromatin stability and in maintaining the structure of sperm tail morphology
(Baccetti et al., 1973). However, exceedingly higher Zn in semen has been linked to Dag defect in bulls (
Blom and Wolstrup, 1976), which augurs well with much high Zn of 984.5 µg/dl in the Ab2. However, the Zn content in seminal plasma of Ab1 resembled to the N bulls. Provision of high dietary Zn also accumulates the element in semen to cause Dag defect (
Chenoweth, 2005). As the feeding strategy / contents were similar for Ab and N, therefore environmental reasons linked to increased dietary Zn causing Dag defect is excluded, which instead might be due to faulty genetics (
Barth and Oko, 1989) requiring cytogenetic confirmation.
Seminal plasma proteins, mainly produced by seminal vesicles
(Westfalewicz et al., 2017), have a multidimensional role in determining the motility and morphology of sperms. Accordingly, a variation in their structure, relative abundance and pattern of expression account for variable semen quality
(Kawano et al., 2004). The electrophoresis generated protein pattern in seminal plasma of the Ab and N bulls is presented in Fig 2. Apparently all the Ab and N bulls shared a homology in the seminal plasma proteins of molecular mass ≥ 30 kDa. However, Ab1 clearly lacked in protein of 15 kDa and also displayed a very weak expression of protein of 25 kDa. The 14 kDA / 15 kDa protein in seminal plasma, known as BSA-A1/A2, is positively correlated to sperm motility, viability
(Bellin et al., 1998) and negatively with the proportion of morphological abnormalities (
Veronica and Puttaswamy, 2000). This is justifiable to some extent in Ab1, whose semen when compared to Ab2, had more viable sperms (35 versus 30%) inspite of which the former exhibited lesser progressively motile sperms (10 versus 15%) and had higher morphological abnormalities (88 and 62%).
It is concluded that the semen with abnormal sperm morphology may also have altered profile of minerals and seminal plasma proteins. Moreover, the latter two evaluations can also be extrapolated in differentiating / justifying otherwise normal ejaculates with varying semen quality parameters.
