<div>Background: 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. 
 
Methods: 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, viz., 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; 
 
Result: 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;m2/s) and dancing mean (&micro;m2/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>

Background: Semen extenders are crucially important when freezing animal semen to protect against sperm cell death and preserve the quality of the sperm cells. Semen diluents play a crucial role in the production process of freezing semen. Both pre-and post-freezing methods can be employed until the point of insemination. 
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Methods: This study aimed to compare the quality of sperm motility after freezing a newly produced semen Extender (MJ Ex2&trade;) with a conventional formula (Tris-egg yolk) using Computer Assisted Sperm Analysis (CASA). This was achieved by dividing the semen into two portions, diluting each portion with a different formula and then freezing both semen specimens using the same process. 
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Result: The results indicate that the fresh goat semen samples in this study had a cream color, an average volume of 1.10&plusmn;0.87 ml/time, a density of 4D (thick), a viscosity of 4oC (milky), a pH of 6.53&plusmn;0.14, a concentration of 17,203&plusmn;275.18 &times; 106/ml and a motility of ++++ (75.7&plusmn;0.13). The MJ Ex2&trade; extender diluent had a movement percentage of 76.72&plusmn;5.00, which was significantly higher than the Tris-egg yolk formula of 66.48&plusmn;2.89&nbsp; (P&lt;0.01). As for the percentage of forward movement, VAP, VSP, ALH and BCF values were not different. The motility characteristics of the Tris-egg Yolk formula had a LIN value of 50.19&plusmn;4.05, which was significantly high (P&lt;0.01). The percentage of motility and STR values of sperm diluted with MJ Ex2&trade; extender was 78.14&plusmn;5.04, which was significantly higher than the Tris-egg yolk formula 72.60&plusmn;7.93 (P&lt;0.05). The results of this study indicate that the MJ Ex2&trade; extender formula enhanced the quality of frozen goat semen. Considering the motility of the sperm; however, would suggest that other effects on those sperm cells would need to be further s to investigate.

Effects of Frozen Semen Extender for Goats (MJ Ex2&trade; and Tris-egg Yolk) on Sperm Motility Quality after Freezing

<div>Background: Taurine supplementation in tris egg yolk citrate (TEYC) extender can improve antioxidant defense and reduce motility degeneration rate in semen of Surti buck preserved at refrigeration temperature. Present study has evaluated antioxidant effect of different concentrations of taurine in TEYC extender on oxidative stress, antioxidant defense and motility degeneration rate in Surti buck semen preserved at refrigerated temperature. 
 
Methods: Four Surti bucks (age&gt;2years) were selected. Total 64 ejaculates (16 per buck) were collected during a period of 8 week. Post-collection they were pooled and stored at refrigeration temperature after dividing into 5 groups based on different taurine concentrations in TEYC extender viz. 0 mM (control T1), 25 mM (T2), 50 mM (T3), 75 mM (T4) and 100 mM (T5) taurine maintaining final concentration of 200 x 106 sperm/ml (pH 6.5-6.8). Evaluation of motility degeneration rate at 24, 36 and 48 hours and glutathione as well as lipid peroxidation (MDA) at 0 and 48 hours was done. 
 
Result: Supplementation of taurine @50 mM in TEYC extender caused post-chilling significant increase in reduced glutathione (GSH) levels, lowering of lipid peroxidation (in terms of MDA production) and reduction of motility degeneration rate (MDR)% in semen of Surti bucks. It improved antioxidant defense thereby maintaining good quality of semen.&nbsp;</div>

​Evaluation of Antioxidant Potential of Taurine in Tris Egg Yolk Citrate Extender in Surti Buck Semen Preserved at Refrigerated Temperature

Background: Intensive use of artificial insemination is the demand of the present era, it deals with cryopreservation. During this process many changes, especially oxidative damages, which affect fertility and can be neutralized by supplementing some antioxidants. 
 
Methods: To conduct this study, a total of thirty-two ejaculates collected from four bulls were preliminary evaluated. Based on progressive motility and viability, qualified samples were processed for cryopreservation. Diluted ejaculates with Egg yolk tris glycerol (EYTG) were split into four aliquots and grouped as Group I: control (without lycopene), Group II, Group III and Group IV as treatment group with 0.25 mM, 0.5 mM and 1 mM lycopene respectively. Post-thaw evaluation for progressive motility, viability, plasma membrane integrity, capacitation status, motility and kinematics, in vitro cervical mucus penetration and antioxidative enzymes activity was performed. 
 
Result: The treatment group containing 0.25 mM lycopene showed significant (P&lt;0.05) improvement in post-thaw sperm progressive motility (%), viability (%), HOS response (%), capacitation status (%), motility and kinematics (CASA), bovine cervical mucus penetration and antioxidant activity (SOD, GST, MDA). This study concluded that lycopene supplementation in bull semen protects spermatozoa from cryo-injury and oxidative stress during the freezing-thawing process. 
 
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Cryoprotective Assessment of Lycopene in Tris-based Extender on Freezability of Bull Semen

<div style="text-align: justify;">Present study was aimed to improve poor quality crossbred bulls semen, as occurrence of poor quality ejaculates are one of the major problems in crossbred bulls. Antioxidants and its combination was tried to overcome such problems. Vitamin E, Vitamin C and Vitamin E+C were supplemented at dose rate of 1mg, 5 mM and 1mg + 5mM per ml respectively in split ejaculates to evaluate semen quality during preservation at refrigerated temperature and cryopreservation. The results showed that semen characteristics were better in antioxidant supplemented ejaculates. Total sperm abnormality were significantly lower (p&lt;0.05) in antioxidant supplemented group. &nbsp;The seminal characteristics of crossbred bull semen showed significantly better (p&lt;0.05) performance on preservation, when fortified with Vitamin E as compared to Vitamin C alone as well as Vitamin E and C in combination. The performance of semen additives, were supportive to semen characteristics especially for Vitamin E, moreover fortification with Vitamin E+C was slightly superior to Vitamin C. It can be concluded that fortification of Vitamin E has beneficial role in semen quality improvement followed by Vitamin E+C and Vitamin C.&nbsp;</div>

Assessment of antioxidants for preservation of crossbred bull semen in Tris based extender

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.

Microbial load of frozen thawed Sahiwal semen extended in egg yolk, soya lecithin and liposome based extender&nbsp;

The aim of this study was to investigate the effects of different extenders viz. Tris citric acid fructose egg yolk (TCFEY), Tris citric acid glucose egg yolk (TCGEY), Egg yolk citrate fructose (EYCF) and Egg yolk citrate glucose (EYCG) on the quality of ram spermatozoa during preservation at 4&deg;C. Semen samples showing more than 3+ mass motility and 70% progressive motility were pooled and subsequently divided into four aliquots. Each aliquot was extended separately in four different extenders viz. TCFEY, TCGEY, EYCF and EYCG and stored at 4&deg;C up to 72h. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in particular extenders (0 h), 24 h, 48 h and 72 h after preservation at 4&deg;C. The percent sperm motility was significantly (P&lt;0.01) higher for TCFEY and TCGEY than EYCF and EYCG at 72 h of preservation at 4&deg;C. The percent HOST reacted spermatozoa and intact acrosomes were significantly (P&lt;0.01) higher and morphological abnormalities were significantly (P&lt;0.01) lower for Tris based fructose extender than other three extenders at 72 h at 4&deg;C. In conclusion, Tris citric acid fructose egg yolk (TCFEY) was found the best in maintaining the quality of ejaculated ram spermatozoa during preservation for 72 h at 4&deg;C. &nbsp;

Effect of different extenders for preservation of ram semen at 4&deg;C

The objective of the present study was to find the comparative efficacy of three extenders to preserve semen of Labrador-Retriever (LR) dogs at 5o C for a short term.&nbsp;&nbsp; &nbsp; The semen samples of LR dogs were collected by digital manipulation method and extended at the rate of 1:4 in Tris-Egg Yolk- Citric Acid-Glucose (TEYCAG), Tris-Egg Yolk- Citric Acid-Fructose (TEYCAF) and Egg Yolk-Citrate-Glycine-Glucose (EYCGG) extenders by split sample technique. &nbsp;Semen was evaluated at 0, 24, 48, 72, 96 and 120 hours of preservation. Mean motile, live and HOST-reacted sperm and acrosomal, head, mid piece and tail abnormalities of spermatozoa varied significantly (P&lt;0.01) between extenders and between preservation periods. The interactions between extender and preservation period were also significant (P&lt;0.01) except for HOST-reacted and head abnormalities of sperm. &nbsp;The highest mean motile, live and HOST-reacted sperm were recorded in TEYCAG extender which did not differ significantly from that of TEYCAF extender. &nbsp;Mean per cent sperm acrosomal and tail abnormalities were significantly (P&lt;0.05) lower, and the incidences of mean sperm head and mid piece abnormalities were also lower in TEYCAG, but not significantly from that in TEYCAF irrespective of hour of preservation. Per cent motile, live and HOST-reacted sperm were significantly (P&lt;0.05) lower and sperm acrosomal, head, mid piece and tail abnormalities were significantly (P&lt;0.05) higher in EYCGG as compared to that in TEYCAG and TEYCAF irrespective of hour of preservation. It was concluded that the semen of LR dog sustained good quality during preservation up to 5 days at 5oC suitable for successful artificial insemination and would be preserved better in TEYCAG and TEYCAF extenders than in EYCGG extender, since more than 50 per cent sperm motility and live sperm were maintained up to 120 hours of preservation in the former two extenders.

Quality of Labrador- Retriever dog semen on short-term preservation in different extenders&nbsp;

The aim of this study was to compare commercially available soybean based extenders, viz. Bioxcell&reg; and Optixcell&reg; (IMV, France) with standard Tris-citrate-fructose-egg yolk-glycerol (TFYG) extender for refrigeration preservation of buffalo semen. Forty eight good quality ejaculates (8/bull) having initial motility &gt;70% obtained at weekly interval in AV from 6 mature Surti buffalo bulls were split-diluted (at 34&deg;C keeping 100&times;106 sperm ml-1) in TFYG, Bioxcell and Optixcell extenders and stored in refrigerator at 5&deg;C for 72 hrs. The mean percentages of progressively motile spermatozoa observed on dilution (0-hr), and after 24, 48, 72 hrs of refrigeration preservation of semen at 5&deg;C were 78.54&plusmn;0.30, 68.30&plusmn;0.30, 59.38&plusmn;0.35 and 51.63&plusmn;0.47; live sperm 90.48&plusmn;0.19, 76.89&plusmn; 0.51, 69.51&plusmn;0.35 and 61.86&plusmn;0.45; morphologically normal sperm 93.85&plusmn;0.08, 90.70&plusmn;0.11, 89.08&plusmn;0.10 and 87.66&plusmn;0.10; intact acrosome 94.40&plusmn; 0.12, 88.22&plusmn;0.13, 84.60&plusmn;0.14 and 81.70&plusmn;0.17, and HOS reactive sperm 79.35 &plusmn;0.24, 65.99&plusmn;0.31, 57.69&plusmn;0.36 and 50.70&plusmn;0.43, respectively, all of which decreased gradually and highly significantly (P&lt;0.01) with increase in storage time. The results were significantly (P&lt;0.05) superior with Optixcell followed by TFYG extender than with the Bioxcell. The overall mean percentages of sperms with abnormal head, mid-piece and tail and those with different forms of acrosomal defects like swollen, ruffled, detached and denuded acrosome also followed the pattern of morphologically normal sperm and intact acrosomes. The values of all these traits increased significantly (P&lt;0.05) with increase in storage duration at 5&deg;C. The sperm protective ability of Optixcell extender was superior followed by TFYG and the least of Bioxcell. The Optixcell, a recently launched improved soybean milk based extender, excelled the Bioxcell extender and was statistically similar to TFYG in respect of all the sperm quality parameters to satisfactorily preserve the quality of buffalo semen at refrigeration temperature up to 3 days. 
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Evaluation of comparative efficacy of egg yolk and soya based extenders&nbsp;for refrigeration preservation (5&ordm;C) of buffalo semen

This study was conducted to develop long-term storage methods for turkey semen using different extenders. During the study, the massage method was used twice a week to collect the semen from five turkeys, a total of 44 times. The collected fresh semen&rsquo;s average ejaculate quantity, sperm concentration, motility and vitality values were determined as 0.22&plusmn;0.01 ml, 3.5&plusmn;0.17 x109 sp/ml, 77.0&plusmn;1.44% and 86.2&plusmn;0.95 % respectively. In this study, glucose (G) including 5% Dimethyl sulphoxide (DMSO) cryoprotective, tris-glucose (TG), lactated Ringer&rsquo;s (LR), and lactated Ringer&rsquo;s glucose (LRG) extenders were used. The Turkey semen was combined and divided into four equal parts, and subsequently diluted at a ratio of 1:3 and equilibrated at +4&deg;C for 90 minutes. Following equilibration, the samples were frozen in liquid nitrogen vapor to &nbsp; - 80&deg;C for five minutes, and stored at -196&deg;C in liquid nitrogen. After freezing and thawing, the highest motility value was obtained from the G extender (43.3% &plusmn;1.62%) followed by LRG (24.6&plusmn;1.53%), LR (12.6&plusmn;0.92%) &nbsp;and &nbsp;TG (12.2&plusmn;0.66%). The vitality values were recorded as 55.8&plusmn;1.89%, 23.8&plusmn;1.58%, 21.5&plusmn;1.10% and 36.5&plusmn;1.59% respectively. The motility and vitality values were significantly (p&lt;0.01) more for the glucose extender than those for other extenders. Therefore, it was concluded that glucose extender is better option for the long-term storage of Turkey semen.

Fortification of Tris Extender with Mifepristone, Sericin and Taurine Improves Velocity and Kinematics of Fresh and Frozen-thawed Bovine Spermatozoa

Effects of Frozen Semen Extender for Goats (MJ Ex2™ and Tris-egg Yolk) on Sperm Motility Quality after Freezing

Evaluation of Antioxidant Potential of Taurine in Tris Egg Yolk Citrate Extender in Surti Buck Semen Preserved at Refrigerated Temperature

Cryoprotective Assessment of Lycopene in Tris-based Extender on Freezability of Bull Semen

Assessment of antioxidants for preservation of crossbred bull semen in Tris based extender

Microbial load of frozen thawed Sahiwal semen extended in egg yolk, soya lecithin and liposome based extender

Effect of different extenders for preservation of ram semen at 4°C

Quality of Labrador- Retriever dog semen on short-term preservation in different extenders

Evaluation of comparative efficacy of egg yolk and soya based extenders for refrigeration preservation (5ºC) of buffalo semen

The effect of different extenders on the sperm motility and viability of frozen Turkey semen