Preliminary Studies of Seminal Attributes and Semen Cryo-banking of Nepali Indigenous Lulu (Bos taurus) Bull under Ex-situ ConservationJha, Pankaj .K; Sapkota, Saroj .; Gurung, Dil .B; Kadel, Raju .; Gorkhali, Neena .A; Shrestha, Bhola .S;
Indigenous animals are valuable genetic property of a country. The Nepali indigenous Lulu cattle breed is in endanger state. Cryopreservation is a simple and potent technology to preserve the gam-etes and could be used for genetic dissemination. Therefore, the study was conducted to evaluate the seminal attributes of fresh semen with a final goal of cryo-banking of Lulu (Bos taurus) semen. Two Lulu bulls were used for the study. A total of 24 ejaculates, 12 ejaculates from each Lulu bull were evaluated for seminal attributes. The mean semen volume, color, mass activity, sperm motility, sperm viability, sperm concentration, abnormal acrosome, mid piece and tail and, abnormal head were 1.6 ± 0.8 ml, 2.8 ± 0.7 grade, 3.1 ± 0.6 grade, 67.5 ± 9.6%, 90.7 ± 4.0%, 977.7 ± 76.1 x 106/ml, 10.1 ± 1.9 and 9.0 ± 1.4%, respectively. Further, 4 ejaculates (≥ 1.5 ml semen volume, ≥ 65% sperm motility and ≥ 800 × 106/ml sperm concentration) from each Lulu bull were used for freezing. The mean mo-tility and viability were 71.9 ± 5.3% and 87.9 ± 6.7% after 90 minutes of cooling. Similarly, mean motility and viability were 70.0 ± 4.6% and 87.1 ± 6.3% after 210 minutes of cooling. The motility and viability of post thawed semen were 51.3 ± 5.2% and 70.4 ± 6.3%. A total number of 174 doses semen straw were cryo-banked. It was concluded that the semen volume, sperm motility and sperm concentration were comparatively lower compared with most of the bulls breed. The sperm viability and abnormal percentage was considered acceptable. Due to the acceptable post thawed sperm motili-ty and viability recorded, cryopreservation of Lulu semen is hereby recommended so as to preserve the Lulu breed. Although, the cryopreserved semen must be tested in fertility trials following artifi-cial insemination.
KeywordsCryopreservation, Nepali indigenous Lulu bull, Semen
Almquist, J.O., Rosenberger, J.L. and Branas, R.J. (1979). Effect of thawing time in warm water on fertility of bovine spermatozoa in plastic straws. Journal of Dairy Science, 62(2): 772-775. doi: https://doi.org/10.3168/jds.S0022-0302(79)83322-6.
Bailey, J.L., Bilodeau, J. and Cormier, N. (2000). Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. Journal of Andrology, 21(1)1-7. doi: 10.1002/j.1939-4640.2000.tb03268.x.
Bhattacharyya, H.K., Goswami, B.K., Bujarbaruah, K.M., Deka, B.C. and Biwas, R.K. (2009). Collection and characterization of semen in Mithun (Bos frontalis) bulls. Theriogenology, 72 (5):699-703. doi: 10.1016/j.theriogenology.2009.05.010.
Bramhaiah, K.V., Rao, V.H., Rao, A.S., Naidu, K.V. and Viroji Rao, S.T. (2013). Semen characteristics of Punganur bulls. Indian Journal of Animal Research, 47(1):45-48.
Chauhan, F.S., Matharoo, J.S., Takkar, O.P. and Singh, M. (1983). Semen characteristics, deep freezing of semen and reproductive performance of crossbred cattle. Indian Journal of Dairy Science, 36(1):96-100.
Colas, G. (1975). Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-freezing ram semen. Journal of Reproduction and Fertility, 42:277-285.
De Lucio, A.C., Ferraudo, A.S., Oliveria de, L.Z., Carno de, A.S., Pereira, W., Carneio, L.C., Lima de, V.F.M.H., Almedia, S., Deragon, L.A., Alves, B.G. and Alves, K.A. (2014). Exploratory analysis of differ-ence in sperm morphology in Nelore and Gir (Bos indicus) bulls. Tropical Animal Health and Production, 46(5):765-770. doi: 10.1007/s11250-014-0561-1.
Dhurvey, M., Gupta, V.K., Nema, S.P., Patidar, A., Shivhare, M., Singh, N. and Shakya, V. (2012). Modern semen evaluation techniques in domestic animals: a review. DHR International Journal of Biomedical and Life Sciences, 3(1):62-83.
and Salvador et al. (2008) reported much lower post thawed viability 44.69 ± 1.00% and 34.50 ± 11.1% in Nellore and Ongole bulls. The re-sult of post thaw semen quality depends upon several factors like type of extender used for processing semen, the amount of glycerol in-cluded in the extender, the rate of cooling used, equilibration period, packaging and rate of freezing and thawing of the semen (Gaillard and Kupferschmied, 1982). The motility and viability of sperm decreased to some extent during cooling is might be due to cold shock, it can be observed as a permanent loss of motility in the thawed sperm. Cold shock usually takes place when the semen temperature rapidly de-creases from approximately body temperature of the bull (35 °C), to the freezing temperature of water (0 °C) (Bailey et al., 2000; Medeiros et al., 2002). The quality of semen deteriorates as the spermatozoa undergoes several ultra-structural, biochemical and functional changes during freezing and thawing (Salamon and Maxwell, 2000).
The semen volume, sperm motility, and sperm concentration were comparatively lower when compared with most of the bull breeds. Due to the acceptable post thawed sperm motility and viability recorded, cryopreservation of Lulu semen is hereby recommended so as to con-serve the Lulu breed. However, the cryo-preserved semen must be tested in fertility tri-als following artificial insemination.
The authors wish to acknowledge the Nepal Agricultural Research Council (NARC) for financial, laboratory facilities and providing the Lulu bulls.
Farooq, U., Ija, A., Ahmad, N., Rehman, H. and Zaneb, H. (2013). Investigation of semen quality and freeza-bility of cholistani breeding bull- A preliminary study from Cholistani desert of Pakistan. The Journal Ani-mal and Plant Science, 23(2):359-363.
Fiaz, M., Usmani, R.H., Abdullah, M. and Ahmad, T. (2010). Evaluation of semen quality of Holstein Friesian and Jersey bulls maintained under subtropical environment. Pakistan Veterinary Journal, 30 (2):75-78.
Fisher, P.S. and Fairfull, R.W. (1984). The effect of glycerol concentration and cooling velocity on cryosurviv-al of ram spermatozoa frozen in straws. Cryobiology, 21(5):542-551. doi: https://doi.org/10.1016/0011-2240(84)90053-1.
Gaillard, C. and Kupferschmied, H. (1982). Thawing time and nonreturn rate of bovine semen frozen in fine French straws. Theriogenology, 18 (4):487-495. doi: https://doi.org/10.1016/0093-691X(82)90170-4
Hafez, E.S.E. (1980). Artificial Insemination. Reproduction in farm animal. Lea and Febriger, Philadelphia, 4th edn. pp 521-45.
Hafez, E.S.E. (1993). Reproduction in farm animals. Lea and Febiger, Philadelphia, 6th edn. pp 405-439.
Haugan, T., Reksen, O., Gröhn, Y.T., Kommisrud, E., Ropstad, E. And Sehested, E. (2005). Seasonal effects of semen collection and artificial insemination on dairy cow conception. Animal Reproduction Science, 90 (1-2):57-71. doi: https://doi.org/10.1016/j.anireprosci.2005.02.002
Jha, P.K., Paul, A.K., Rahman, M.B., Tanjim, M., Bari, F.Y. and Alam, M.G.S. (2013). Improvement of preservation quality of chilled bull semen using α-tocopherol as an antioxidant. Journal of Embryo Trans-fer, 28 (1):31-39. doi: http://dx.doi.org/10.12750/JET.2013.28.1.31.
Joshi, B.R., Shrestha, B.S., Kadel, R., Gorkhali, N., Sapkota, S. and Manandhar, H.K. (2015). Participatory conservation of Lulu cattle: A case study for an unique cattle breed on Nepal Himalayas. Symposium on the strategies for the promotion of result diffusion of AFASI projects, Jeonju, Korea, pp.33-46.
Kedia, N.K., Tiwari, R.P., Mishra, G.K., Poyam, M.R., Pandey, A.K., Nair, A.K. and Sahasrabhude, S.A. (2014). Characteristics and Freezability of Tharparkar bull semen. Indian Journal of Animal Sciences, 84 (4):382-388.
Lagerlof, N. (1934). Morphological studies on the change in the sperm structure and in the testis of bulla with decreased or abolished fertility. Acta Pathology Microbiology Scandinavica, 19: p254.
Medeiros, C.M., Forell, F., Oliveira, A.T. and Rodrigues, J.L. (2002). Current status of sperm cryopreserva-tion: why isn’t it better? Theriogenology, 57 (1):327-344. doi: https://doi.org/10.1016/S0093-691X(01)00674-4
Mishra, S.R., Kundu, A.K. and Mahapatra, A.P.K. (2013). Effect of ambient temperature on membrane integri-ty of spermatozoa in different breeds of bulls. The Bioscan, 8 (1):181-183.
Naing, S.W., Haron, A.W., Khan, M.A. and San, M.M. (2011). Effect of seminal plasma removal, washing solutions, and centrifugation regimes on Boer goat semen cryopreservation. Pertanika Journal of Tropical Agricultural Science, 34(2):271-279.
Nath, K.C., Ahmed, K., Dutta, G., Barthakur, T. and Borgohain, B.N. (1996). Semen quality and release of certain enzymes during the course of freezing bull spermatozoa. Indian Journal of Animal Reproduction, 17:130-131.
Neopane, P.N. and Pokharel, P.K. (2005). Indigenous cattle of Nepal. Animal Breeding Division, National Animal Science Research Institute (NASRI), Nepal Agricultural Research Council (NARC), Khumaltar, Lalitpur, Kathmandu, Nepal, p2-8.
Neopane, S.P., Kunwar, B.S. and Jhoshi, H.D. (2002). Characterization of Lulu cattle in high mountains of Nepal. Paper presented at the International Seminar on Mountains organized by Royal Nepal Academy of Science and Technology, Kathmandu, Nepal.
Patel, B.R. and Siddiquee, G.M. (2013). Physical and morphological characteristics of Kankrej bull semen. Veterinary World, 6 (7):405-408. doi: 10.5455/vetworld.2013.405-408
Pontbriand, D., Howard, J.G., Schiewe, M.C., Stuart, L.D. and Wildt, D.E. (1989). Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa. Cryobiolo-gy, 26 (4):341-354. doi: https://doi.org/10.1016/0011-2240(89)90058-8
Ramukhithi, F.V., Nedambale, T.L., Sutherland, B. and Lehloenya, K.C. (2011). Cryopreservation of South African indigenous goat semen. African Journal of Biotechnology, 10 (77):17898-17902. doi: 10.5897/
Rana, C.M. and Dhami, A.J. (2004). Physical attributes, intact acrosome, HOS test and freezability of semen of Gir and Jafarabadi bulls. Indian Veterinary Journal, 81:401-410.
Salamon, S. and Maxwell, W.M. (2000). Storage of ram semen. Animal Reproduction Science, 62 (1-3):77-111. doi: https://doi.org/10.1016/S0378-4320(00)00155-X.
Salvador, D.F., Andrade, V.J., Vale Filho, V.R., Dias, J.C. and Nogueira, L.A.G. (2008). Relationship between andrologic profile and semen freezing in two year old Nellore bulls, pre select by breeding soundness eval-uation. Arquio Brasilerio de Medicina Veterinária Zootechnia, 60 (3):587-593. doi: http://dx.doi.org/10.1590/S0102-09352008000300010.
Siddiqui, M.A.R., Bhattacharjee, J., Das, Z.C., Islam, M.M., Islam, M.A., Haque, M.A., Parrish, J.J. and Shamsudin, M. (2008). Cross bred bull selection for bigger scrotum and shorter age at puberty with poten-tials for better quality semen. Reproduction in Domestic Animal, 43 (1):74-79. doi: 10.1111/j.1439-0531.2007.00857.x
Singh, D.M. and Pangawkar, G.R. (1990). Studies on semen characteristics of exotic and crossbred bulls sper-matozoa. Indian Journal of Animal Reproduction, 11:92-95.
Sugulle, A.H., Bhuiyan, M.M.U. and Shamsuddin, M. (2006). Breeding soundness of bull and the quality of their frozen semen used in cattle artificial insemination in Bangladesh. Livestock Research for Rural Devel-opment, 18(4):54.
Talluri, T.R., Rao, M.M., Rao, K.B. and Naidu, K.V. (2011). Semen ejaculate characteristics and fertility in Ongole bulls. Indian Veterinary Journal, 88(2):36-37.
Veeraiah, G., Venugopal, K. and Rao, K.B. (1999). Effect of various level of glycerol and egg yolk in freezing of Ongole bull semen. Indian Journal of Animal Reproduction, 20 (2):149-152.
Verstegen, J., Iguer-Ouada, M. and Onclin, K. (2002). Computer assisted semen analyzers in andrology re-search and veterinary practice. Theriogenology, 57(1):149-179. doi: https://doi.org/10.1016/S0093-691X(01)00664-1.
Vilakazi, D.M. and Webb, E.C. (2004). Effect of age and season on sperm morphology of Friesland bulls at an artificial insemination center in South Africa. South African Journal of Animal Science, 34 (1):62-69. doi: http://dx.doi.org/10.4314/sajas.v34i1.4041.