<p>Sixty semen ejaculates from 10 mature bulls, 5 each of Jafarabadi and Mehsana breed, were studied for sperm motility and velocity parameters of fresh and frozen-thawed spermatozoa using computer assisted sperm analyzer (CASA). The mean values of motile and progressively motile spermatozoa observed in fresh semen of Jafarabadi and Mehsana bulls (79.77&plusmn;1.62 and 61.80&plusmn;1.85, and 78.90&plusmn;1.22 and 61.37&plusmn;1.58%) were highly significantly (Pand medium motile sperm (%) increased, while beat-cross frequency (Hz), straightness (%), linearity (%), sperm area (&micro;m2) and rapidly motile sperm (%) decreased significantly in post-thawed sperms when compared with the fresh sperm of both Jafarabadi and Mehsana bulls. The initial motility and live sperm per cent were significantly correlated with CASA traits of fresh and frozen-thawed semen, and all the sperm motility and velocity traits of fresh and frozen-thawed semen assessed by CASA were significantly interrelated among both the breeds. The interrelationships were stronger in Mehsana bulls as compared to Jafarabadi bulls.</p>


<p>Computer assisted sperm analysis of fresh and frozen-thawed buffalo semen and their interrelationship</p>


<div><strong>Background:</strong> The quality of the frozen thawed semen is one of the most powerful factor affecting the conception rate in a buffaloes. The season may have its effect on post thaw seminal attributes and fertility. Therefore, current study was aimed to investigate the effect of winter and summer seasons on post thaw seminal attributes and conception rate from semen of Jaffarabadi buffalo bulls.<br />
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<strong>Methods: </strong>The study was conducted on four Jaffarabadi breeding bulls, 5-6 years old, to know the post-thaw semen characteristics based on total of 192 semen ejaculates evaluated and cryopreserved over one year period.&nbsp;<br />
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<strong>Result: </strong>The mean percentages of post-thaw motility, live sperm, abnormal sperm, HOST reactive sperm, acrosomal integrity and first AI conception rate for semen cryopreserved during winter were 58.85&plusmn;0.56, 67.06&plusmn;0.77, 8.62&plusmn;0.43, 58.52&plusmn;0.67, 75.22&plusmn;0.27 and 45.75&plusmn;0.21, respectively, whereas, corresponding values for semen cryopreserved during summer were 56.35&plusmn;0.43, 65.62&plusmn;0.76, 9.08&plusmn;0.51, 55.43&plusmn;0.60, 74.29&plusmn;0.22 and 43.50&plusmn;0.11. The semen quality of post thawed samples were observed better during winter compared to summer season for artificial insemination with satisfactory first AI conception rate.</div>


<div>Seasonal Influence on Post Thaw Seminal Attributes and Conception Rate from Semen of Jaffarabadi Buffalo Bulls</div>


<div><strong>Background:</strong> The evaluation of bull fertility essentially involves semen analysis and conception rates among inseminated females. The conventional evaluation is relatively imprecise, time consuming and subjected to individual variations, while computer assisted sperm analysis (CASA) is a quite faster, precise and objective tool to identify differences in sperm motility and velocity/kinematics attributes and avoids subjective errors. The present study was planned to evaluate and compare the CASA traits of fresh and frozen-thawed semen of Gir and Murrah bulls in Tris extender without and with different antioxidant additives by adopting Biovis CASA.<br />
<br />
<strong>Methods: </strong>The semen ejaculates with &gt;75% initial motility from 3 Gir and 3 Murrah bulls were split-diluted at 100 million sperm per ml using Tris-citrate-fructose-yolk-glycerol (TFYG) extender without (control) and with three antioxidant additives, <em>viz</em>., Mifepriston (10 &micro;g/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml), were filled in French mini straws and frozen in liquid nitrogen vapour using a programmable biofreezer. The freshly diluted and frozen-thawed semen samples were assessed for sperm motion characteristics, velocity/kinematics using Biovis CASA.&nbsp;<br />
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<strong>Result:</strong> The mean percentages of total motile spermatozoa, irrespective of additives, in freshly diluted and frozen-thawed semen were 85.50&plusmn;0.92 and 48.76&plusmn;1.69 for Gir bulls and 85.03&plusmn;0.72 and 52.61&plusmn;1.46 for Murrah bulls, respectively. The mean total motile as well as rapid and slow progressive motile sperm percentage were significantly enhanced by fortification of TFYG extender with Mifepristone than in control extender, while values for Sericin and Taurine were intermediary and statistically similar to Mifepristone. The per cent decline in total motile sperm due to freezing-thawing was more or less same in both the breeds for all the extender additives, being lowest in Mifepristone supplemented extender in cattle (32.27 vs 48.81%) and buffalo (31.27 vs 43.14%) semen. The rapid and slow progressive motile sperm also followed the same pattern. The values of VAP and VCL were significantly (p&lt;0.05) higher and VSL lower in Murrah than Gir breed at post-thaw stage. The overall mean linearity (%), straightness (%), beat-cross frequency (hz), lateral head displacement (&micro;m), wobbling index (%), dancing velocity (&micro;m<sup>2</sup>/s) and dancing mean (&micro;m<sup>2</sup>/s) of sperm were higher in Murrahs than in Gir semen with significant (p&lt;0.01) difference at post-thaw stage. The fortification of extender with Mifepristone improved all these traits at post-thaw stage compared to other additives and control TFYG extender. The per cent decline in these traits due to freezing stress was much lower in buffalo sperm than the cattle sperm particularly with Mifepristone fortification. It was concluded that freezing-thawing stress adversely affected the motion and kinematics of both cattle and buffalo sperm and that Tris extender fortified with antioxidant additives, particularly Mifepristone @ 10 &micro;g/ml, protected sperm from adverse effect of dilution and cryopreservation with improved post-thaw sperm quality.</div>


<div>Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa&nbsp;</div>


<div style="text-align: justify;">A study was undertaken on semen of three mature bulls each of Gir, Surti and Murrah breed to evaluate the comparative motion characteristics and kinematics of their fresh and frozen-thawed spermatozoa by Biovis CASA. The ejaculates (n= 24/breed) having &gt;75% initial motility were diluted @ 80 million sperm/ml using TFYG extender and were assessed. Amongst motility traits, the total motile, rapid progressive motile and slow progressive motile spermatozoa percentage decreased significantly by 23.08 - 30.09, 43.57 - 55.18, 9.12 - 22.75 %, respectively, (p&lt;0.01), while non-progressive motile sperm (4.78 - 21.48%) and immotile sperm (164.38 - 178.38 %) percentage increased significantly (p&lt;0.01) in frozen-thawed semen compared to that of fresh semen. The post-thaw quality of semen of all three breeds was in acceptable range. The mean values of sperm velocity/kinematic parameters observed in frozen-thawed semen of Gir, Surti and Murrah bulls, based on total motile sperms, viz., average path velocity, &nbsp;curvilinear velocity, straight line velocity, linearity, straightness, beat-cross frequency, amplitude of lateral head displacement, dancing frequency and dancing mean decreased significantly by 13.70 - 17.79; 9.76 - 12.95; 13.28 - 21.90; 7.28 - 9.68; 4.36 - 7.79; 15.56 - 25.15; 8.78 - 10.50; 6.16 - 18.67 and 12.98 - 15.96 %, respectively, as compared to that of their fresh semen samples. However, wobbling index remained almost same for both fresh and frozen semen. All motility traits differed but none of kinematics/velocity traits differed significantly between breeds/species. The values of all velocity parameters for progressive motile sperms were higher than total motile sperms in all three breeds. The effect of freezing-thawing on velocity and kinematic attributes was much less compared to absolute sperm motility, and both the fresh and frozen-thawed sperms behaved identically with respect to their velocity and kinematics. The rapid progressive motile sperm in both fresh (r=0.41 to 0.92) and frozen-thawed (r=044 to 0.88) semen had significant correlations with most of their velocity traits, and the later were significantly and positively or negatively inter-related among each other in semen of all three breeds. It was therefore concluded that cryopreservation process significantly reduces the motility and kinematics attributes of bovine spermatozoa and, CASA analysis of fresh semen for motility and velocity traits could predict the post-thawed sperm motility and velocity/ kinematics of spermatozoa.</div>

<div style="text-align: justify;">Comparative Evaluation of Motility and Kinematics of Fresh Versus Frozen-Thawed Spermatozoa of Cattle and Buffalo Bull by CASA</div>

<p>The objective of this study was to investigate the effect of using different extenders viz. Androhep, Safecell Plus and SUS during cryopreservation on quality of frozen-thawed boar semen. Semen volume, sperm motility, sperm concentration, percentage of morphologically abnormal spermatozoa, total number of spermatozoa per ejaculate and activity of the enzyme aspartate aminotransferase (AST) were assessed in fresh semen collected from 39 fertile AI boars. Semen from each boar was divided into three portions and diluted 1:1.5 in extender Androhep, Safecell Plus and SUS and keep at 17&deg;C for 15-h holding time before cryopreservation. Then sperm was cryopreserved. Straws were thawed in a water bath at 38&deg;C for 40s and post-thaw sperm motility with AST activity was assessed. &nbsp;Significant difference in post-thaw sperm motility was found between extender Androhep and Safecell Plus (P&lt;0.05). AST activity did not differ significantly between tested extenders (P&gt;0.05). In conclusion, the results of the study indicate that using Safecell Plus extender during holding period before cryopreservation significantly affected post-thaw sperm motility.</p>


<p>Effect of different extenders on quality of frozen-thawed boar semen</p>


<div style="text-align: justify;">Study was conducted to compare the semen quality and fertility of liquid stored semen for three days and frozen-thawed semen in the north-eastern region of India. For liquid semen, the semen ejaculates were extended in Beltsville Thawing Solution (BTS) extender and preserved at 17&deg;C for three days. For cryopreservation, semen was diluted Lactose-egg yolk-glycerol extender and frozen in straw using programmable freezer with freezing rate of 40&deg;C/min from -6 to -140&deg;C. The preserved evaluated for sperm motility, viability, plasma membrane integrity and fertility. The results revealed that the liquid stored semen has maintained the sperm motility and viability up to day 3 without significant reduction. Similarly the plasma membrane integrity did not differ significantly up to day 2, but it was significantly (P&lt;0.05) reduced on days 3 in liquid stored semen. After freezing and thawing, the mean sperm motility, viability and plasma membrane integrity were 58.25 &plusmn; 2.96%, 64.75 &plusmn; 2.47% and 47.06 &plusmn; 2.02%, respectively. These parameters were significantly (P&lt;0.01) lower as compared to the liquid stored semen from day 0 to day 3. After insemination with liquid semen, the farrowing rate was 77.7%, 80.76%, 73.07% and 69.8%, respectively from day 0, day1, day 2 and day 3. The pregnancy rate, farrowing rate and litter size did not differ significantly among different days of liquid storage. These parameters were significantly (P&lt;0.01) lower in frozen semen as compare to that of liquid stored semen. The study concluded that the liquid semen stored up to three days is more efficient than frozen-thawed semen in terms of preserving sperm quality and fertility.</div>

<div style="text-align: justify;">Semen quality and fertility of liquid stored and frozen-thawed semen in crossbred pigs of North-Eastern India</div>

<div style="text-align: justify;">Goats are important livestock species of India. Chegu is a pashmina producing goat native to the cold arid region of Himachal Pradesh (H.P.), India. Semen cryopreservation from six Chegu elite males (aged 2.05&plusmn;0.40 years; weighed 29.16&plusmn;2.02 kg) was practiced using Tris Citrate Egg Yolk extender containing 10% EY and 6% Glycerol. Gross semen parameters includes volume (0.80.85&plusmn;0.07 ml), color (Creamy white to yellowish), concentration (2238.5&plusmn;231.0 x106 spermatozoa/ml) and mass motility (3.92&plusmn;0.03).The significant changes (P&lt;0.01) in post thaw seminal parameters (75.48&plusmn;0.69 v/s 37.38&plusmn;0.90; progressive motility), viability (75.79&plusmn;0.95 v/s 48.25&plusmn;1.78), morphological abnormalities (5.64&plusmn;0.29 v/s 7.02&plusmn;0.32) and HOST reactive spermatozoa (64.07&plusmn;1.75 v/s 43.35&plusmn;1.79) were observed in present study. Artificial insemination using frozen thawed semen having concentration (150 x106 spermatozoa/straw) from three different bucks was practiced in 40 synchronized goats with conception rate of 42.5 per cent. Non-significant variations amongst different bucks were observed with birth of 1.12 kids per doe and twinning rate of 11.8 per cent. It was concluded that semen cryopreservation along with artificial insemination can be practiced in Chegu goats to improve the population of this endangered species.&nbsp;</div>

Cryopreservation and fertility of frozen thawed Chegu goat semen

<div><strong>Background:</strong> Pantja is a medium sized, dual purpose Tarai goat breed of Uttarakhand and adjacent area of Uttar Pradesh. Due to the lack of pure breeding buck and uncontrolled cross breeding, population of this breed is reducing continuously. Therefore, for preservation and dissemination of this pure breed, cryopreservation of semen and artificial insemination by using frozen thawed semen can play a crucial role.&nbsp;<br />
<br />
<strong>Methods:</strong> Semen samples were collected from four sexually matured Pantja bucks and after preliminary evaluation extended in tris egg yolk based extender. Post thaw semen was examined for microscopical and certain biochemical evaluation. A total 12 does were inseminated and pregnancy diagnosis was conducted at 45 days onwards by ultrasonography.&nbsp;<br />
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<strong>Result:</strong>&nbsp;Post thaw seminal attributes were significantly (P&pound;0.01) reduced in comparision to freshly diluted semen. Total six does (50.00%) were pregnant and five (83.33%) of them gave birth to healthy kids. Among these five, three does (60.00%) gave birth to single and two does (40.00%) gave birth to twins.</div>

<div style="text-align:justify">&nbsp;</div>


<div>In vivo Fertility Assessment of Frozen Thawed Pantja Buck Semen</div>


<div><strong>Background:</strong> Oxidative stress occurs when oxygen or oxygen derived oxidants exceed antioxidants and become responsible for poor post-thaw semen quality during the cryopreservation process. Therefore, the present study was designed to investigate the effects of dissolved oxygen (DO) levels (4, 6 and 8 ppm) in semen extender on freezability of crossbred bull spermatozoa. As the gap between 4 and 8 ppm existed in earlier studies, DO of 6 ppm in semen extender was further standardized and the effects were compared with other respective groups to contemplate any improvement in post-thaw semen quality.<br />
<br />
<strong>Methods:</strong> For the experiment, Tris-egg Yolk-Glycerol (TYG) extender was partially deoxygenated by nitrogen gassing @ 2-3 bubbles/sec at 34&deg;C for 0, 16, 12 and 9 minutes to obtain DO levels of 11.7 ppm (Group-I/control), 4 ppm (Group-II), 6 ppm (Group-III) and 8 ppm (Group IV), respectively and collected semen samples were diluted with these extender groups to have 80&times;10<sup>6</sup> spermatozoa/ ml of the extender. Semen samples were evaluated for individual progressive motility (IPM), lipid peroxidation (LPO), total antioxidant capacity (TAC), plasma membrane integrity, acrosomal integrity and apoptotic changes at different stages of cryopreservation.<br />
<br />
<strong>Result: </strong>At the post-thaw stage, progressive motility was greater (p&lt;0.05) in Group II compared to Group I and the least reduction from the post-dilution to the post-thaw stage was observed in Group II. In comparison to Group I, Groups II, III and IV showed lesser (p&lt;0.05) MDA production with Group II having greater (p&lt;0.05) TAC concentration than other groups at the post-thaw stage. A declining trend was observed in membrane integrity as DO levels increased from 4 ppm to 11.7 ppm. Acrosomal integrity did not differ among treatment groups, but, found to be higher (p&lt;0.05) than the control group. Per cent viable spermatozoa was greater (p&lt;0.05) in Group II than Group I and vice versa for necrotic spermatozoa as assessed by Annexin V<sup>FITC</sup>/PI staining. In conclusion, reducing the DO level to 4 ppm before cryopreservation improved the freezability by reducing oxidative stress and apoptotic changes while, above 4 ppm tended to lower it. An appreciable improvement in freezability can be seen at 6 ppm of DO, but, not up to that extent as observed at 4 ppm.</div>


<div>Partial Deoxygenation of Semen Extender Minimizes Post-thaw Damages and Improves Freezability of Crossbred Bull Spermatozoa</div>


<p style="text-align: justify;">Present investigation was carried out to study the bacterial load of different semen extender in frozen thawed semen samples of Sahiwal bulls maintained at Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana. Twenty four ejaculates from four bulls were collected during morning hours using sterilized artificial vagina and the ejaculates were evaluated. Semen samples showing more than 70 per cent progressive motility and having more than 800 million sperms per ml were used for further processing and freezing. The samples were split into six aliquots and extended in six different extenders [Conventional egg yolk (CEY), egg yolk of hen supplemented with omega enriched diet (group I), Egg yolk of hen supplemented with herbal feed (group II), 1% soya lecithin (SY1), commercially available Bioxcell (SY2) and Optixcell (LP)]. One ml of post thaw semen (four straws) was serially diluted (ten-fold) with normal saline, loaded in sterilized nutrient agar plate and incubated at 37&deg;C for 48 hours. Number of colonies were counted in duplicate and multiplied by dilution factor. The Standard plate counts (CFU/ml) of frozen thawed semen samples extended in different extenders were analyzed by one way analysis of variance. The results revealed that bacterial load of SY1, SY2 and LP was significantly (p&lt;0.05) different from egg yolk based extenders. The highest microbial load of frozen thawed semen samples was recorded in group II (646.30&plusmn;71.65) egg yolk extender and lowest in optixcell (91.67&plusmn;6.72) extender. Microbial load in all the extenders are well within the standard limit (5000CFU/ml) of frozen thawed semen sample. Lower bacterial load in present finding may be due to maintenance of better HACCP protocol for hygiene during preparation, semen collection and semen processing.</p>
