Mukul Anand

Assessment of motion and kinematic characteristics of frozen-thawed Sirohi goat semen using computer-assisted semen analysis.

Aim: The aim was to determine the motion and kinematics characteristic of frozen-thawed spermatozoa in Sirohi goat using computer-assisted semen analysis. Materials and Methods: A study was carried out in Sirohi buck. Semen collection was made biweekly from each buck with the help of artificial vagina. A total of 12 ejaculates were collected from two bucks (six ejaculates from each buck). Freshly collected semen was pooled and later evaluated. The pooled semen sample was extended with standard glycerolated egg yolk tris extender and later subjected to a process of cryopreservation. The motion and kinematic characteristics of spermatozoa were studied during freez-thawing process. Results: Significantly (p<0.01) higher value of live percent, hypo-osmotic swelling test, and acrosomal integrity were recorded in neat semen followed by diluted and frozen thaw semen. The proportion of spermatozoa showing slow progression were the highest in the neat and diluted semen followed by rapid and non-progressively motile, while a reverse pattern was observed in the frozen thaw semen where the proportion of non-progressively motile spermatozoa were significantly (p<0.01) higher followed by slow and rapid progression. Conclusion: This study showed that the best results for motion, vitality, plasma membrane integrity, and acrosome status were obtained in the neat semen followed by diluted and frozen thaw semen. Further, the process of cryopreservation results in a shift of motility from slow to non-progressive in the post-thaw semen with a significant decrease in the path velocities when compared to neat and diluted semen. Hence, it can be concluded that freezing-thawing process reduces the motility and kinematic characters spermatozoa and may be an important factor affecting the fertilizing ability of spermatozoa resulting in poor conception rate after insemination in goats.

Effect of egg yolk concentration and washing on sperm quality following cryopreservation in Barbari buck semen

ABSTRACT The present study was conducted to evaluate the effect of egg yolk concentration and washing on sperm quality in cryopreserved Barbari buck semen at higher dilution (100 × 106 cells/ml). Five healthy Barbari bucks of similar age and weight were selected as semen donor. Six ejaculates were collected from each buck during the experiment. Collected semen samples were pooled and diluted with Tris-based semen extender containing 6% glycerol and egg yolk according to the treatments – T-1, that is, 3% egg yolk, T-2, that is, 20% egg yolk and T-3, that is, 20% egg yolk after washing and cryopreserved using the slow freezing technique. Semen was evaluated at three different steps, namely, after dilution, after equilibration and after freeze–thawing. Significantly (p ≤ .01) higher values for motility and viability were observed in T-3 just after dilution while significantly (p ≤ .01) higher values for acrosomal integrity and pattern F (uncapacitated sperm) was observed in T-2. Significantly (p ≤ .01) higher values were observed in T-2 after equilibration and thawing. It may be concluded that incorporation of 20% egg yolk compared to 3% egg yolk or 20% egg yolk after washing in extender confer better cryoprotection to Barbari buck spermatozoa as reflected in semen quality parameters studied.

Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa

Present study was undertaken to characterize the voltage gated potassium channel (Kv 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 μL diluted sperm samples namely-negative control (100 μL of sperm dilution medium (SDM) containing 10 × 106 cells), vehicle control (99 μL of SDM containing 10 × 106 cells, and DMSO- 1  μL) and 4-AP treatment group (99 μL of SDM containing 10 × 106 cells, and drug 1 μL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies are warranted to unravel the mechanistic signaling pathways involved in Kv-mediated alterations in functional dynamics of spermatozoa.