Much controversy surrounds the clinical significance of an increased concentration of white blood cells (WBC) in the male ejaculate. The World Health Organization's classification of leukocytospermia is a concentration > 1 x 10(6) WBC/ml. The aim of this study was to assess the association of varying concentrations of leukocytes to sperm morphology evaluated by strict criteria. Semen samples were collected from a total of 79 patients. Round cells on the initial semen analysis were stained for identification of polymorphonuclear granulocytes (PMN) as the largest group (50-60%) of white blood cells using the Endtz Method commercially produced as Leucoscreen. Diff Quick Staining Kit was used for sperm morphology assessment and 200 spermatozoa were assessed per slide. Data were evaluated using two cut-off criteria, at 0.5 x 10(6) WBC/ml and 1 x 10(6) WBC/ml. Mann-Whitney U-values at both < and > 0.5 x 10(6)/ml PMN (P < 0.001) and at < and > 1.0 x 10(6)/ml PMN (P < 0.015) showed differences between percentage normal forms. Spearman's rank correlation coefficient for PMN concentration showed a negative correlation (P < 0.01) with percentage normal sperm morphology and positive correlation for midpiece abnormalities (P < 0.04). These data support the hypothesis that PMN have a role in the increase of abnormal spermatozoa, particularly those with midpiece abnormalities, by as yet unknown mechanisms.

. How to cite this article: Thomas J, Singh M, Goswami TK, Verma S, Badasara SK (2014). Polymerase chain reaction based epidemiological investigation of canine parvoviral disease in dogs at Bareilly region, Veterinary World 7(11): 929-932. AbstractAim: The aim of this study was to screen the suspected samples by polymerase chain reaction (PCR) and epidemiological analysis of positive cases of canine parvovirus type2. Materials and Methods:Fecal samples were collected from dogs suspected for canine parvovirus type 2 (CPV-2) and viral DNA was extracted. Primers were designed, and PCR was done with all extracted DNA samples. Age, sex and breed wise distribution of positive cases were analyzed.Results: Out of a total 44 collected fecal samples, 23 were found to be positive for CPV-2 by developed PCR. The disease was found to be more common in Labrador male pups of 3-6 months of age. The percentage of positive cases in vaccinated dogs was found to be around 17.4%. Conclusion:Almost half (52.3%) of total collected samples were found to be positive by PCR. However, number of field samples are needed to further validate this test and additionally sequence analysis needs to be done to ensure the prevalent field strain of CPV-2.

Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

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Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 μg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 μg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 μM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.