الفهرس 
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Abstract
The present study was carried out at Animal Production Research Station, Sakha, Kafrelsheikh Governorate, APRI, ARC, Ministry of Agriculture, Egypt, in cooperation with Physiology and Biotechnology Laboratory, Department of Animal Production, Faculty of Agriculture, Mansoura University, Egypt, during the period from August, 2016 until February, 2017. This study aimed to evaluate the effect of alternative components of egg yolk (SBL or BHT) in Tris-extender and semen packaging methods (straws or pellets) during cryopreservation. 5 Ossimi and 5 Finnish rams aged 2-4 years for semen collection once weekly for 28 weeks using artificial vaginal (140 ejaculates for each breed). Only semen ejaculates with mass motility of ≥70% were pooled, divided and diluted with three extender types. Tris-citric-egg yolk extender (Tris-EY) with 15% fresh egg yolk was used as a control extender. Egg yolk in Tris-EY was replaced by 1% soybean lecithin (Tris-SBL) or 2 mM butylated hydroxy toluene (Tris-BHT). Pooled semen was diluted at a ratio of 1:5 (semen/extender). Semen was placed for cooling in the refrigerator (5oC) for 4 hours as equilibration period. Equilibrated semen was frozen liquid nitrogen at -196°C by two methods of packaging, 0.25 ml French straws and 0.25 ml pellets. Semen thawed at 37°C for 30 s in a water bath to evaluate sperm characteristics. For both Ossimi and Finnish semen, progressive motility, livability, abnormality and curled tail spermatozoa were evaluated in post-diluted, post-equilibrated and post-thawed semen. Sperm DNA damage, sperm apoptosis and sperm ultrastructure using TEM were also evaluated. Concentration of TAC, MDA and LDH activity were determined in post-thawed seminal plasma. Fertility of Ossimi frozen-thawed semen was evaluated using 40 mature crossbred ewes (½ Ossimi x ½ Finish Landrace), having 3-4 years. Ewes were artificially inseminated at 48-56 h after CIDR withdrawal. The results of the present study could be summarized as follows: 1. Effect of extender type on sperm characteristics : 1.1. In post-diluted Ossimi and Finnish semen: Sperm characteristics were not affected significantly by the type of extender. 1.2. In post-equilibrated Ossimi and Finnish semen: Only percentage of curled tail spermatozoa in post-equilibrated Ossimi semen was higher (P<0.05) in Tris-EY and Tris-SBL extenders than in Tris-BHT extender. 2. Sperm characteristics as affected by extender type, packaging method and their interaction in post-thawed Ossimi and Finnish semen: 2.1 Effect of extender type: Overall mean of all sperm characteristics in post-thawed semen were nearly similar in Ossimi and Finnish semen. 2.2. Effect of packaging methods: Overall mean of sperm progressive motility, livability and curled tail sperm percentages in post-thawed Ossimi Finnish rams semen improved in straws than in pellets. Overall mean of sperm abnormality percentage was not affected in both ram breeds. 2.3. Effect of interaction: Effect of interaction on all sperm characteristics was not significant. 3. Antioxidants capacity and LDH activity in post-thawed seminal plasma of Ossimi and Finnish rams : 3.1. Effect of type of extender: Overall mean of TAC in seminal plasma of Ossimi semen was not affected significantly by extender type, but it was the highest (P<0.05) in Tris-BHT of Finnish seminal plasma and the lowest in Tris-EY. Overall mean of MDA concentration in both breeds was the highest (P<0.001) in Tris-EY seminal plasma and the lowest in Tris-BHT. Overall mean of LDH activity was the highest (P<0.001) in Tris-BHT seminal plasma of Ossimi semen and the lowest in Tris-EY, but was not affected significantly by extender type in Finnish seminal plasma. 3.2. Effect of semen packaging method : Only overall mean of MDA concentration in seminal plasma of Ossimi rams was higher (P<0.05) in pellets than in straws. Overall mean of TAC, LDH activity in Ossimi, TAC, MDA concentration and LDH activity in Finnish showed different trend in each breed with insignificant differences between straw and pelleted semen. 3.3. Effect of the interaction: Effect of interaction was significant (P<0.01) only on LDH activity in seminal plasma of Ossimi rams. This effect reflected in the highest LDH activity of Tris-BHT semen in Ossimi. 4. Sperm flow cytometer in post-thawed Ossimi semen:4.1. Effect of extender type: Overall mean of viable and early apoptotic sperm percentages were the highest (P<0.001), while overall mean of apoptotic sperm percentage was the lowest (P<0.05) in Ossimi semen extended with Tris-EY extender. 4.2. Effect of packaging method: Overall mean of viable was higher (P<0.001), necrotic sperm percentages was higher (P<0.05) and percentages of early apoptotic and apoptotic spermatozoa was lower (P<0.001) when Ossimi semen was packaged in straws than in pelleted form. 4.3. Effect of interaction: Viable and necrotic sperm percentages were higher in straw than in pelleted semen for all extender types, being the highest in Ossimi semen diluted with Tris-EY in straw semen. 5. Comet assay of post-thawed Ossimi semen : 5.1. Effect of extender type: Tris-EY extender decreased (P<0.001) percentage of DNA Damage as compared to Tris-SBL or Tris-BHT extenders. This trend was observed for tail length, percentage of DNA in tail and tail moment, but the differences were not significant.5.2. Effect of packaging method: Straws decreased all comet assay parameters as compared to pelleted semen.5.3. Effect of the interaction: The significant effect on DNA damage percentage (P<0.001) and tail length (P<0.01), reflected lower DNA damage in straws than in pellets only for semen diluted with Tris-EY or Tri-SBL. 6. Sperm ultrastructure (Transmission Electron Microscope, TEM): Many spermatozoa with obvious heads with normal nuclei, acrosomal membrane and plasma membrane, and mid, principle and end pieces with normal plasma membrane and axonome with all types of extenders and semen packaging methods. Sperm abnormalities in terms of disturbances and disintegration of plasma membrane, swelling plasma membrane and completely lysis of plasma membrane, loose acrosome, higher atypical acrosome and acrosome reaction, and abnormal nuclei, distorted mitochondria and slightly deformation of axonome were more detectable in pelleted than in straw semen. 7. Fertility study: Overall mean of pregnancy rate was the highest for Tris-BHT semen, moderate for Tris-SBL semen and the lowest for Tris-EY semen, but the differences were not significant. Overall mean of litter size/ewe was higher for Tris-SBL semen than for Tris-EY and Tris-BHT. 7.2. Effect of packaging method: Overall mean of pregnancy rate and litter size was higher for straws semen than for pelleted semen. 7.3. Effect of the interaction: Pregnancy rate was not significant, reflecting higher pregnancy rate of semen diluted with Tris-BHT as compared to other extender types, being the highest for pelleted semen. CONCLUSION The successful usage of Tris-SBL or Tris-BHT, without adversely effects on sperm characteristics, in comparing with the possible disadvantages of using egg yolk, including its potential to be a cause of allergic reactions, the risk of bacterial contamination and its variable effect on semen, which may allow the save using SBL or BHT as an alternative of egg yolk in Tris-based extender of ram semen. Ossimi semen extended with Tris-SBL in straws showed the highest fertility rate, followed by those extended with Tris-BHT.