Effect of time and depth of insemination on fertility of Bharat Merino sheep inseminated trans-cervical with frozen-thawed semen

  • Kumar, Davendra (Division of Animal Physiology and Biochemistry, Central Sheep and Wool Research Institute) ;
  • Naqvi, Syed Mohammed Khursheed (Division of Animal Physiology and Biochemistry, Central Sheep and Wool Research Institute)
  • Received : 2014.05.05
  • Accepted : 2014.07.17
  • Published : 2014.07.31


Background: Artificial insemination (AI) can serve as a powerful tool to the sheep owners for making rapid genetic progress of their flock. The AI in sheep is mostly performed using fresh semen with two reasons i) lambing rate following trans-cervical AI with frozen semen is limited by the inability of frozen-thawed sperm to transit the cervix and ii) the need of circumventing the cervical barrier through laparoscope aided intrauterine AI. Therefore, AI with frozen-thawed semen is not as widespread in sheep as it is in other domestic species. However, to get maximum benefits through the use of AI, frozen-thawed semen is a prerequisite because instead of high fertility, the short shelf life of fresh semen coupled with a limitation on the number of insemination doses achievable per unit time restricts the widespread use of individual sires. Therefore, in order to enhance lambing rate, a total of 240 trans-cervical artificial inseminations with frozen-thawed semen were performed in Bharat Merino ewes during autumn season either once in the evening (G-I, 10 h after onset of estrus, n = 100) or twice (G-II, 14 h and 22 h after onset of estrus, n = 140) i.e. once in the morning and again in the evening. Results: The pregnancy rate (proportion of pregnant ewes confirmed by ultrasonography at day 40) and lambing rate (proportion of ewes lambed) were higher in G-II as compared to G-I (26.4 vs 20% and 19.3 vs 10%, respectively). The difference in lambing rates was statistically (P < 0.05) significant. The depth of insemination within cervico-uterine tract had no significant effect on pregnancy and lambing rates. Conclusions: The results indicate that lambing rate in sheep following TCAI with frozen-thawed semen was significantly influenced by time of inseminations. Two inseminations after 14 and 22 h of onset of estrus enhanced the lambing rates of Bharat Merino sheep as compare to single insemination after 10 h of onset of estrus. The TCAI technique with frozen-thawed ram semen is promising and may serve as a valuable tool for genetic improvement of sheep breeds. Research efforts are going on worldwide to overcome the poor fertility following TCAI with frozen-thawed semen.


  1. Maxwell WMC, Hewitt LJ: A comparison of vaginal, cervical and intrauterine insemination of sheep. J Agricul Sci 1986, 106:191-193.
  2. Halbert GW, Dobson H, Walton JS, Buckrell BC: The structure of the cervical canal of the ewes. Theriogenology 1990, 33:977-992.
  3. Naqvi SMK, Pandey GK, Guatam KK, Joshi A, Geethalaxmi V, Mittal JP: Evaluation of gross anatomical features of cervix of tropical sheep using cervical silicone moulds. Anim Reprod Sci 2005, 85:337-344.
  4. Anel L, Alvarez M, Martinez-Pastor F, Garcia-Macias V, Anel E, Paz P: Improvement strategies in ovine artificial insemination. Reprod Domestic Anim 2006, 41:30-42.
  5. Salamon S, Maxwell WMC: Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim Reprod Sci 1995, 46:89-96.
  6. Naqvi SMK, Joshi A, Mathur AK, Bag S, Mittal JP: Intrauterine artificial insemination of Malpura ewes in natural estrus with frozen ram semen. Ind J Anim Sci 1997, 67:982-983.
  7. Fukui Y, Kohno H, Togari T, Hiwasa M, Okabe K: Fertility after artificial insemination using a soybean-based semen extender in sheep. J Reprod Develop 2008, 54:286-289.
  8. Aisen EG, Alvarez HL, Venutrino A, Garde JJ: Effect of trehalose and EDTA on cryoprotective action of ram semen diluents. Theriogenology 2000, 53:1053-1061.
  9. Buckrell BC, Buschbeck C, Gartlet CJ, Kroetsch T, McCutcheon W, Martin J, Penner WK, Walton JS: A breeding trial using a transcervical technique for artificial insemination in sheep. Proc 12th Int Cong Anim Reprod 1992, 3:1531-1533.
  10. Windsor DP, Szell AZ, Buschbeck C, Edward AY, Milton JTB, Buckrell BC: Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen. Theriogenology 1994, 42:147-157.
  11. Buckrell BC, Buschbeck C, Gartley CJ, Kroetsch T, McCutcheon W, Martin J, Penner WK, Walton JS: Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen. Theriogenology 1994, 42:601-611.
  12. Naqvi SMK, Joshi A, Bag S, Pareek SR, Mittal JP: Cervical penetration and transcervical AI of tropical sheep (Malpura) at natural estrus using frozen-thawed semen. Technical Note. Small Rum Res 1998, 29:329-333.
  13. Cappai P, Sanna SR, Branca A, Fraghi A, Bomboi G: Comparison of laparoscopic and transcervical insemination with frozen semen in Sarda dairy ewes. Anim Sci 1998, 66:369-373.
  14. Wulster-Radcliffe MC, Wang S, Lewis GS: Transcervical artificial insemination in sheep: effect of new transcervical artificial insemination instrument and traversing the cervix on pregnancy and lambing rates. Theriogenology 2004, 62:990-1002.
  15. Gunduz MC, Turna O, Cirit U, Ucmak M, Tek C, Sabuncu A, Bacinoglu S: Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes. Anim Reprod Sci 2010, 118:32-36.
  16. Alvarez M, Chamorro CA, Kaabi M, Anel-Lopez L, Boixo JC, Anel E, Anel L, de Paz P: Design and "in vivo" evaluation of two adapted catheters for intrauterine transcervical insemination in sheep. Anim Reprod Sci 2012, 131:153-159.
  17. Campbell JW, Harvey TG, McDonald MF, Sparksman RI: Transcervical insemination in sheep: an anatomical and histological evaluation. Theriogenology 1996, 45:1535-1544.
  18. Wulster-Radcliffe MC, Lewis GS: Development of a new transcervical artificial insemination method for sheep: effects of a new transcervical artificial insemination catheter and traversing the cervix on semen quality and fertility. Theriogenology 2002, 58:1361-1371.
  19. Salamon S, Maxwell WMC: Storage of ram semen. Anim Reprod Sci 2000, 62:77-111.
  20. Park JE, Graham JK: Effects of cryopreservation procedures on sperm membranes. Theriogenology 1992, 38:209-222.
  21. Salamon S: Fertility following deposition of equal numbers of frozen-thawed ram spermatozoa by single and double insemination. Aust J Agri Res 1977, 28:477-479.
  22. Kershaw C, Khalid M, McGowan M, Ingram K, Leethongdee S, Wax G, Scaramuzzi R: The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen. Theriogenology 2005, 64:1225-1235.
  23. Halbert GW, Dobson H, Walton JS, Buckrell BC: A technique for transcervical intrauterine insemination of ewes. Theriogenology 1990, 33:993-1010.
  24. Kaabi M, Alvarez M, Anel E, Chamorro CA, Boixo JC, de Paz P, Anel L: Influence of breed and age on morphometry and depth of inseminating catheter penetration in the ewe cervix: a postmortem study. Theriogenology 2006, 66:1876-1883.
  25. Eppleston J, Maxwell WMC: Recent attempts to improve the fertility of frozen ram semen inseminated into the cervix. Wool Tech Sheep Breed 1993, 41:291-302.
  26. Eppleston J, Salamon S, Moore NW, Evans G: The depth of cervical insemination and site of intrauterine insemination and their relationship to the fertility of frozen-thawed ram semen. Anim Reprod Sci 1994, 36:211-225.
  27. Paulenz H, Soderquist L, Adnoy T, Nordsroga A, Berg KA: Effect of vaginal and cervical deposition of semen on the fertility of sheep inseminated with frozen-thawed semen. Vet Rec 2005, 156:372-375.
  28. Szabados T, Gergatz E, Vitinger E, Zi T, Gyoker E: Lambing rate as a function of artificial insemination depth in ewe lambs, primiparous and multiparous ewes. Acta Agraria Kaposvariensis 2005, 9:41-49.
  29. Leethongdee S: Development of trans-cervical artificial Insemination in sheep with special reference to anatomy of cervix. Suranaree J Sci Technol 2010, 17:57-69.
  30. Taqueda GS, Azevedo HC, Santos EM, Matos JE, Bittencourt RF, Bicudo SD: Influence of anatomical and technical aspects on fertility rate based on sheep transcervical Artificial insemination performance. ARS Veterinaria 2011, 27:127-133.
  31. Richardson L, Hanrahan JP, Donovan A, Marti JI, Fair S, Evans AC, Lonergan P: Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen. Anim Reprod Sci 2012, 131:160-164.
  32. Kahn W, Achtzehn J, Kahn B, Richter A, Schulz J, Wolf M: Sonography of pregnancy in sheep. II Accuracy of transrectal and transcutaneous pregnancy diagnosis. Dtsch Tierarztl Wochenschr 1993, 100:29-31.
  33. Yotov S: Diagnostics of early pregnancy in Stara Zagora dairy sheep breed. Bulg J Vet Med 2005, 8:41-45.
  34. Dixon AB, Knights M, Winkler JL, Marsh DJ, Pate JL, Wilson ME, Dailey RA, Seidel G, Inskeep EK: Patterns of late embryonic and fetal mortality and association with several factors in sheep. J Anim Sci 2007, 85:1274-1284.
  35. Langford GA, Marcus GJ, Hackett IJ, Ainsworth L, Wolynetz MS, Peters HF: A comparison of fresh and frozen semen in the insemination of confined sheep. Canad J Anim Sci 1979, 59:685-691.
  36. Olesen I: Effects of cervical insemination with frozen semen on fertility and litter size of Norwegian sheep. Livestock Prod Sci 1993, 37:169-184.
  37. Singh VK, Arora AL, Mehta BS, Mishra AK, Kumar S, Prince LLL: Bharat Merino. In New Strains of Sheep Evolved. Avikanagar (India): CSWRI; 2006:21.
  38. Schmehl MK, Anderson SP, Vazques IA, Graham EF: The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility. Cryobiology 1986, 23:406-414.
  39. Joshi A, Kumar D, Naqvi SMK, Maurya VP: Effect of controlled-rate cooling and freezing on motion characteristics and acrosomal integrity of cryopreserved ram spermatozoa. Cell Pres Tech 2008, 6:277-284.
  40. Snedecor GW, Cochran WG: Statistical Methods. 8th edition. Ames, Iowa: Iowa State University Press; 1989.