REVIEW_ARTICLE Artificial insemination (AI) as a part of assisted reproductive technologies represents the oldest and most widespread method used to accelerate genetic progress in all domestic animals. After its first implementation in ovine reproduction and almost 80 years afterward, AI is continuously used for improving the genetic merit, utilizing either fresh or shorttime chilled semen. Nevertheless, regardless of the semen used for insemination, the conception rate (CR) is still lower in comparison to natural service. At least two factors are commonly thought to limit the success of the AI and reduce the CR: (1) failure of placing the semen directly into the uterus due to the specific anatomic structure of the ewe’s cervix; (2) lower viability of ram spermatozoa during cryopreservation ( < 30% progressively motile spermatozoa after thawing). This review elaborates on recent studies that aimed to achieve acceptable CR through the implementation of cervical or intrauterine insemination: deep intracervical, intrauterine trans-cervical, and intracornual. Several hormonal treatments (oxytocin, estrogen, or prostaglandin) were evaluated on inducing cervical dilation that facilitates insemination. A comprehensive analysis was given to the effects of several antioxidants (GSSG, GSH, and cysteine) supplemented in ram semen-freezing media. Sex-sorted ram semen fertility rate results were presented from our studies. https://macvetrev.mk/Files/Article/2022/10.2478/macvetrev-2022-0018/macvetrev-2022-0018.pdf 2022-10-15 109 125 10.2478/macvetrev-2022-0018 artificial insemination sheep insemination ejaculate conservation sex sorted semen Toni Dovenski dovenski@fvm.ukim.edu.mk false 1 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia LEAD_AUTHOR Plamen Trojacanec false 2 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Branko Atanasov false 3 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Martin Nikolovski false 4 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Vladimir Petkov false 5 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Florina Popovska-Percinic false 6 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Monika Dovenska false 7 Faculty of Veterinary Medicine-Skopje, Ss Cyril and Methodius University, Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Juraj Grizelj false 8 Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia AUTHOR Silvijo Vince false 9 Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia AUTHOR Nenadović, K., Karać, P., Vučinić, M., Teodorović, R., Živanov, D., Trailović, R., Beckei, Z., Janković, Lj. (2020). Assessment of the welfare of extensively managed autochthonous sheep breed Vlasicka Yackel using animal-based measurements. Acta Vet. 70(2): 207-218. 1 10.2478/acve-2020-0015 Barillet, F.S., Sanna, D., Boichard, J.M., Astruc, A., Carta, S., Casu. (1993). Genetic evaluation of the Lacaune, Manech and Sarda dairy sheep with animal model. Proceedings of 5th International Symposium on Machine Milking of Small Ruminants, May, 14-20, (pp. 289-303), Budapest, Hungary 2 Mikuš, T., Kozačinski, L., Cvrtila, Ž., Vince, S., Špoljarić, B., Perkov, S., Kardum Paro, M.M., Špoljarić, D., Pajurin, L., Popović, M. (2021). The influence of season and age on the levels of lipids, lipoproteins and enzymes in the serum of Lička pramenka sheep. Veterinarski arhiv 91(1): 31-37. 3 10.24099/vet.arhiv.0954 Jakovljević, G., Lazarević, M., Mirilović, M., Milovanović, A., Apić, J., Šefer, D., Nedić, S., Vakanjac, S. (2021). The influence of zinc and heavy metals in feed and water on the quality of cryopreserved bull semen. Acta Vet. 71(3): 326-343. 4 10.2478/acve-2021-0028 Woelders, H., Zuidberg, C.A., Sulkens, H., Pieterse, M., Peterson, K., Hiemstra, S.J. (2005). Cryopreservation and insemination of ejaculated and epididymal semen from Dutch rare sheep breeds. Proceedings of 56th Annual Meeting of the European Association for Animal Production Vol. 11, June, 5-8, (pp. 282), Uppsala, Sweden 5 Ehling, C., Rath, D., Struckmann, C., Frenzel, A., Schindler, L., Niemann, H. (2006). Utilization of frozen-thawed epididymal ram semen to preserve genetic diversity in Scrapie susceptible sheep breeds. Theriogenology 66(9): 2160-2164. PMid:16905182 6 10.1016/j.theriogenology.2006.07.003 Woelders, H., Windig, J., Hiemstra, S. (2012). How developments in cryobiology, reproductive technologies and conservation genomics could shape gene banking strategies for (farm) animals. Reprod Domest Anim. 47, 264-273. PMid:22827380 7 10.1111/j.1439-0531.2012.02085.x Nikolovski, M., Atanasov, B., Dovenska, M., Petkov, V., Dovenski, T. (2013). Variations in semen quality parameters of Ovchepolian Pramenka rams according to the method of collection and the meteorological season. Mac Vet Rev. 35(2): 79-86. 8 Ledesma, A., Manes, J., Cesari, A., Alberio, R., Hozbor, F. (2014). Electroejaculation increases low molecular weight proteins in seminal plasma modifying sperm quality in corriedalerams. Reprod Domest Anim. 49(2): 324-332. PMid:24494601 9 10.1111/rda.12279 Wulster-Radcliffe, M.C., Wang, S., Lewis, G.S. (2004). Transcervical artificial insemination in sheep: effects of a new transcervical artificial insemination instrument and traversing the cervix on pregnancy and lambing rates. Theriogenology 62(6): 990-1002. doi:10.1016/j.theriogenology.2003.12.031 PMid:15289042 10 10.1016/j.theriogenology.2003.12.031 Graham, J. K. (2001). Assessment of sperm quality: a flow cytometric approach. Anim Reprod Sci. 68(3-4): 239-247. doi:10.1016/s0378-4320(01)00160-9 11 10.1016/S0378-4320(01)00160-9 Gadea, J., Matás, C., Lucas, X. (1998). Prediction of porcine semen fertility by homologous in vitro penetration (hIVP) assay. Anim Reprod Sci. 54(2): 95-108. 12 10.1016/S0378-4320(98)00144-4 Ivanova, M., Mollova, M. (1993). Zona-penetration in vitro test for evaluating boar sperm fertility. Theriogenology 40(2): 397-410. doi:10.1016/0093-691x(93)90277-c 13 10.1016/0093-691X(93)90277-C Januskauskas, A., Gil, J., Söderquist, L., Hrd, M.G.M., Hrd, M.C., Johannisson, A., Rodriguez-Martinez, H. (1999). Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in sweden. Theriogenology 52(4): 641-658. 14 10.1016/S0093-691X(99)00159-4 Fazeli, A.R., Zhang, B.R., Steenweg, W., Larsson, B., Bevers, M.M., Van Den Broek, J., Rodriguez-Martinez, H., Colenbrander, B. (1997). Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen. Theriogenology 48(5): 853-863. 15 10.1016/S0093-691X(97)00310-5 Trojacanec, P., Kocoski, Lj., Dovenski, T., Popovski, K., Petkov, V., Mickovski, G., Trojacanec, S. (1999). Prediction of in vitro fertilization capacity of bulls semen after ionofore-induced acrosome reaction. Proceedings of the 3rd ESDAR Conference, November, (Abstr. 90), Anger, France 16 Nikolovski, M., Dovenska, M., Ilievska, K., Adamov, N., Atanasov, B., Radeski, M., Kirovski, D., Petkov, V., Dovenski, T. (2019). Homologous seminal plasma and glutathione promote pre-capacitation motility and structural stability of cryopreserved ram spermatozoa. Mac Vet Rev. 42(2): 169-179. 17 10.2478/macvetrev-2019-0022 Hancock, J.L. (1951). A staining technique for the study of temperature-shock in semen. Nature 167(4243): 323-324. PMid:14806490 18 10.1038/167323b0 Rijsselaere, T., Van Soom, A., Maes, D., Nizanski, W. (2012), Computer-assisted sperm analysis in dogs and cats: an update after 20 years. Reprod Domest Anim. 47, 204-207. PMid:23279500 19 10.1111/rda.12057 Trojacanec, P., Kocoski, L., Dovenski, T., Petkov, V., Popovski, K., Trojacanec, S., Andonov, S. (2000). In vitro fertilization potential correlates with mechanical and functional integrity of the sperm plasma membrane. 14th Int. Congr. Anim. Reprod., July, 2-6, (abst. 2:15), Stockholm, Sweden 20 Ramu, S., Jeyendran, R.S. (2012). The hypo-osmotic swelling test for evaluation of sperm membrane integrity. In D. Carrell, K. Aston (Eds.), Spermatogenesis. Methods in molecular biology (Methods and protocols, vol. 927 (pp. 21-25). Totowa, NJ: Humana Press PMid:22992900 21 10.1007/978-1-62703-038-0_3 Galarza, D.A., López-Sebastián, A., Woelders, H., Blesbois, E., Santiago-Moreno, J. (2018). Sephadex filtration as successful alternative to density-gradient centrifugation procedures for ram sperm selection with improved kinetics. Anim Reprod Sci. 192, 261-270. PMid:29580753 22 10.1016/j.anireprosci.2018.03.022 Ahmad, Z., Anzar, M., Shahab, M., Ahmad, N., Andrabi, S.M. (2003). Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates. Theriogenology 59(5-6): 1189-1202. 23 10.1016/S0093-691X(02)01159-7 Cunha, A.T.M., Carvalho, J.O., Dode, M.A.N. (2015). Techniques for sperm evaluation using fluorescent probes. Semina: Ciênc Agrár. 36(6Supl2): 4365. 24 10.5433/1679-0359.2015v36n6Supl2p4365 Zeginiadou, T., Papadimas, J., Mantalenakis, S. (2000). Acrosome reaction: methods for detection and clinical significance. Andrologia 32(6): 335-343. PMid:11131842 25 10.1046/j.1439-0272.2000.00359.x Mocé, E., Graham, J.K. (2008). In vitro evaluation of sperm quality. Anim Reprod Sci. 105(1-2) : 104-118. PMid:18178345 26 10.1016/j.anireprosci.2007.11.016 Ferrari, S., Barnabe, V.H., Züge, R.M., Zogno, M.A. (2000). Effect of two ram sperm capacitating media on acrosome reaction and zona-free hamster oocyte penetration test. BJVRAS 37, 229-233. 27 10.1590/S1413-95962000000300010 Evenson, D.P. (1999). Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 14(4): 1039-1049. PMid:10221239 28 10.1093/humrep/14.4.1039 Evenson, D.P., Larson, K.L., Jost, L.K. (2006). Sperm chromatin structure assay: its clinical use for setecting aperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl. 23(1): 25-43. PMid:11780920 29 10.1002/j.1939-4640.2002.tb02599.x Robayo, I., Montenegro, V., Valdés, C., Cox, J. (2008). CASA assessment of kinematic parameters of ram spermatozoa and their relationship to migration efficiency in ruminant cervical mucus. Reprod Domest Anim. 43(4): 393-399. PMid:18282216 30 10.1111/j.1439-0531.2007.00920.x Shukla, M.K., Misra, A.K. (2007). Effect of bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation. Anim Reprod Sci. 97(1-2) : 175-179. PMid:16600533 31 10.1016/j.anireprosci.2006.02.015 Larsson, B., Rodrı́guez-Martı́nez, H. (2000). Can we use in vitro fertilization tests to predict semen fertility? Anim Reprod Sci. 60-61: 327-336. 32 10.1016/S0378-4320(00)00089-0 Emamverdi, M., Zhandi, M., Zare Shahneh, A., Sharafi, M., Akbari-Sharif, A. (2013). Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender. Reprod Domest Anim. 48(6): 899-904. PMid:23701189 33 10.1111/rda.12183 Chemineau, P., Cagnie, Y., Guerin, Y., Orgeur, P., Vallet, J.C. (1991). Training manual on artificial insemination in sheep and goats (No. FAO APHP-83). FAO, Roma (Italia) [in French] 34 Evans, G, Maxwell, W.M.C. (1987) Salamon's artificial insemination of sheep and goats. Sydney, NSW: University Press, Butterworth-Heinemann 35 Martínez-Soto, J.C., Landeras, J., Gadea, J. (2012). Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology 1(3): 365-375. PMid:23596043 36 10.1111/j.2047-2927.2012.00040.x Bailey, J.L., Blodeau, J.F., Cormier, N. (2000), Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon minireview. J Androl. 21, 1-7. 37 Nikolovski, M., Dovenska, M., Uzunov, R., Hajrulai-Musliu, Z., Petkov, V., Dovenski, T. (2014). Change in fatty acid composition of chilled ram semen in relation to spermatozoa motile activity. Mac Vet Rev. 37 (Suppl. 1): 32. 38 10.14432/j.macvetrev.2014.05.014 Salamon, S., Maxwell, W.M. (2000). Storage of ram semen. Anim Reprod Sci. 62(1-3): 77-111. 39 10.1016/S0378-4320(00)00155-X Nikolovski, M., Mickov, Lj., Dovenska, M., Petkov, V., Atanasov, B., Dovenski, T. (2014). Influence of glutathione on kinetic parameters of frozen-thawed spermatozoa from Ovchepolian Pramenka rams, Mac Vet Rev 2014. 37(2): 121-128. 40 10.14432/j.macvetrev.2014.05.014 Bucak, M.N., Ateşşahin, A., Yüce, A. (2008). Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rumin Res. 75(2-3): 128-134. 41 10.1016/j.smallrumres.2007.09.002 Halbert, G.W., Dobson, H., Walton, J.S., Buckrell, B.C. (1990). The structure of the cervical canal of the ewe. Theriogenology 33(5): 977-992. 42 10.1016/0093-691X(90)90060-7 Kershaw, C.M., Khalid, M., McGowan, M.R., Ingram, K., Leethongdee, S., Wax, G., Scaramuzzi, R.J. (2005). The anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen. Theriogenology 64(5): 1225-1235. PMid:15904956 43 10.1016/j.theriogenology.2005.02.017 Halbert, G.W., Dobson, H., Walton, J.S., Buckrell, B.C. (1990). A technique for transcervical intrauterine insemination of ewes. Theriogenology 33(5): 993-1010. 44 10.1016/0093-691X(90)90061-W Perry, K., Haresign, W., Wathes, D.C., Khalid, M. (2010). Intracervical application of hyaluronan improves cervical relaxation in the ewe. Theriogenology 74(9): 1685-1690. PMid:20833422 45 10.1016/j.theriogenology.2010.07.008 Gündüz, M.C., Turna, Ö., Cirit, Ü., Uçmak, M., Tek, Ç., Sabuncu, A., Bacınoğlu, S. (2010). Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes. Anim Reprod Sci. 118(1): 32-36. PMid:19586730 46 10.1016/j.anireprosci.2009.06.001 Candappa, I.B.R., Bainbridge, H.C., Price, N.T., Hourigan, K.R., Bartlewski, P.M. (2009). A preliminary study on the suitability of Cervidil® to induce cervical dilation for artificial insemination in ewes. Res Vet Sci. 87(2): 204-206. PMid:19303615 47 10.1016/j.rvsc.2009.02.004 Leethongdee, S., Khalid, M., Bhatti, A., Ponglowhapan, S., Kershaw, C.M., Scaramuzzi, R.J. (2007). The effects of the prostaglandin E analogue Misoprostol and follicle-stimulating hormone on cervical penetrability in ewes during the peri-ovulatory period. Theriogenology 67(4): 767-777. PMid:17126896 48 10.1016/j.theriogenology.2006.10.012 Dovenski, T., Trojacanec, P., Petkov, V., Popovska-Percinic, F., Kochoski, L., Grizelj, J. (2012). Laparoscopy-promising tool for improvement of reproductive efficiency of small ruminants. Mac Vet Rev. 35(1): 5-11. 49 Milovanović, A., Maksimović, N., Barna, T., Lazarević, M., Delić, N. (2013). Laparoscopic insemination of sheep in Republic of Serbia. Biotechnol Anim Husb. 29(3): 449-456. 50 10.2298/BAH1303449M Kocoski, Lj., Popovski, K., Georgievski, B., Dovenski, T., Mickovski, G., Petkov, V., Trojacanec, P. (1991). Introduction of laparascopic technique in sheep and goats. Proceedings of Summer Conference for Sheep and Goat Production (pp. 125-129), Ohrid, Macedonia [in Macedonian] 51 Dovenski, T., Popovski, K., Petkov, V., Mickovski, G., Kocoski, L., Trojacanec, P., Stojanovski, B. (1997). Comparison of intrauterine and cervical insemination in goats with deep-frozen semen. Proceedings of 1st Croatian Veterinary Congress (pp. 279-285), October, Cavtat, Croatia 52 Milovanović, A., Milovanović, B., Barna, T., Lazarević, M., Aleksijević, D. (2012). First laparoscopic insemination of sheep with deep - frozen semen in the Republic of Serbia.Scientific Symposium Reproduction of Domestic Animals and Diseases of Newobrns. October, 4-7, (pp. 143-148), Divčibare, Serbia 53 Croy, B.A., Prudencio, J., Minhas, K., Ashkar, A.A., Galligan, C., Foster, R.A., Buckrell, B., Coomber, B.L. (1999). A preliminary study on the usefulness of huil-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer. Theriogenology 52(2): 271-287. 54 10.1016/S0093-691X(99)00128-4 Khalifa, R.M., Sayre, B.L., Lewis, G.S. (1992). Exogenous oxytocin dilates the cervix in ewes. J Anim Sci. 70(1): 38-42. PMid:1582916 55 10.2527/1992.70138x Sayre, B.L., Lewis, G.S. (1997). Fertility and ovum fertilization rate after laparoscopic or transcervical intrauterine artificial insemination of oxytocin-treated ewes. Theriogenology 48(2): 267-275. 56 10.1016/S0093-691X(97)84074-5 Shabankareh, H.K., Sarsaifi, K., Mehrannia, T. (2011). In vitro maturation of ovine oocytes using different maturation media: effect of human menopausal serum. J Assist Reprod Genet. 28, 531-537. PMid:21152965 PMCid:PMC3158248 57 10.1007/s10815-010-9523-3 Mardenli, O., Al-Kerwi, M.S.M., Alolo, A.Y. (2021). The effect of follicle size and cryoprotectants on nuclear maturation and early embryonic development of vitrified-thawed Awassi sheep oocytes (Ovis aries). Veterinarski Arhiv 91(5): 483-493. 58 10.24099/vet.arhiv.1063 Paramio, M.T., Izquierdo, D. (2014). Current status of in vitro embryo production in sheep and goats. Reprod Domest Anim. 49, 37-48. PMid:25277431 59 10.1111/rda.12334 Paramio, M.T. (2010). In vivo and in vitro embryo production in goats. Small Rumin Res. 89, 144-148. 60 10.1016/j.smallrumres.2009.12.037 de Matos, D.G., Gasparrini, B., Pasqualini, S.R., Jeremy, G.T. (2002). Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology 57, 1443-1451. 61 10.1016/S0093-691X(02)00643-X Shabankareh, H.K., Kafilzadeh, F., Soltani, L. (2012). Treatment of ovine oocytes with certain water-soluble vitamins during in vitro maturation (IVM). Small Rumin Res 104, 139-145. 62 10.1016/j.smallrumres.2011.09.050 Catala, M.G., Izquierdo, D., Rodriguez-Prado, M., Hammami, S., Paramio, M.T. (2012). Effect of oocyte quality on blastocyst development after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in a sheep model. Fertil Steril. 97, 1004-1008. PMid:22265000 63 10.1016/j.fertnstert.2011.12.043 Guler, A., Poulin, N., Mermillod, P., Terqui, M., Cognie, Y. (2000). Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes. Theriogenology 54, 209-218. 64 10.1016/S0093-691X(00)00342-3 Wani, A.R., Khan, M.Z., Sofi, K.A., Lone, F.A., Malik, A.A., Bhat, F.A. (2012). Effect of cysteamine and epidermal growth factor (EGF) supplementation in maturation medium on in vitro maturation, fertilization and culturing of embryos in sheep. Small Rumin Res. 106, 160-164. 65 10.1016/j.smallrumres.2012.02.015 Tibary, A., Anouassi, A., Khatir, H. (2005). Update on reproductive biotechnologies in small ruminants and camelids. Theriogenology 64, 618-638. PMid:15964064 66 10.1016/j.theriogenology.2005.05.016 Cocero, M., Alabart, J., Hammami, S., Marti, J., Lahoz, B., Sanchez, P., Echegoyen, E., Beckers, J., Folch, J. (2011). The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte. Reprod Domest Anim. 46, 463-470. PMid:20887399 67 10.1111/j.1439-0531.2010.01690.x Shabankareh, H.K., Zandi, M. (2010). Developmental potential of sheep oocytes cultured in different maturation media: effects of epidermal growth factor, insulin-like growth factor I, and cysteamine. Fertil Steril. 94, 335-340. PMid:19324348 68 10.1016/j.fertnstert.2009.01.160 Shirazi, A., Shams-Esfandabadi, N., Ahmadi, E., Heidari, B. (2010). Effects of growth hormone on nuclear maturation of ovine oocytes and subsequent embryo development. Reprod Dom Anim. 45, 530-536. PMid:19032427 69 10.1111/j.1439-0531.2008.01290.x Souza-Fabjan, J.M.G., Locatelli, Y., Duffard, N., Corbin, E., Touze, J.L., Perreau, C., Beckers, J.F., Freitas, V.J.F., Mermillod, P. (2014). In vitro embryo production in goats: slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media. Theriogenology 81, 1021-1031. PMid:24582267 70 10.1016/j.theriogenology.2014.01.023 Rizos, D., Ward, F., Duffy, P., Boland, M., Lonergan, P. (2002). Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 61, 234-248. PMid:11803560 71 10.1002/mrd.1153 Shirazi, A., Motaghi, E. (2013). The in vitro fertilization of ovine oocytes in the presence of oviductal cells and its effect on the expression of zygote arrest 1 (zar1) and subsequent embryonic development. J Reprod Infertil. 14, 8-16. 72 Heidari, B., Shirazi, A., Naderi, M.M., Akhondi, M.M., Hassanpour, H., Sarvari, A., Borjian, S. (2013). Effect of various co-culture systems on embryo development in ovine. Czech J Anim Sci. 58, 443-452. 73 10.17221/6993-CJAS Wang, Z., Lin, P., Yu, S. (2013). Effects of ghrelin on developmental competence and gene expression of in vitro fertilized ovine embryos. Theriogenology 79, 695-701. PMid:23290751 74 10.1016/j.theriogenology.2012.11.026 Rho, G., Hahnel, A., Betteridge, K. (2001). Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro. Theriogenology 56, 503-516. 75 10.1016/S0093-691X(01)00581-7 Lahoz, B. (2013). Influence of the FecX+R++ allele in heterozygous ewes on follicular population and outcomes of IVP and ET using LOPU-derived oocytes. Reprod Dom Anim. 48, 717-723. PMid:23438026 76 10.1111/rda.12150 Sreenivas, D., Thomas, V.M., Kaladhar, D.S.V.G.K., Yarla, N.S., Palni Samy, A., Varahalarao, V., Preethi, R. (2013). In vitro production of sheep embryos in CR1aa medium supplemented with L-ascorbic acid: effects on morula and blastocyst development. J Bioanal Biomed. 5, 187-193. 77 10.4172/1948-593X.1000097 Urdaneta, A., Jimenez, A., Paramio, M., Izquierdo, D. (2004). Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes. Zygote 12, 277-284. PMid:15751535 78 10.1017/S0967199404002874 Shabankareh, H.K., Akhondi, M. (2012) Effect of glucose levels and MEM vitamins during the first step of IVC in synthetic oviduct fluid medium on in vitro development of bovine zygotes. Small Rumin Res. 106, 54-58. 79 10.1016/j.smallrumres.2012.04.023 Cox. J.F., Alfaro, V. (2007). In vitro fertilization and development of OPU derived goat and sheep oocytes. Reprod Dom Anim. 42, 83-87. PMid:17214779 80 10.1111/j.1439-0531.2006.00735.x Wan, P.C., Hao, Z.D., Zhou, P., Wu, Y., Yang, L., Cui, M.S., Liu, S.R., Zeng, S.M. (2009). Effects of SOF and CR1 media on developmental competence and cell apoptosis of ovine in vitro fertilization embryos. Anim Reprod Sci. 114, 279-288. PMid:19008058 81 10.1016/j.anireprosci.2008.09.020 Li, F., Pi, W.H., Zhua, H.Z., Zhang, S.S., Liu, S.R., Xue, J.L. (2006). The effect of estrous ewe serum and heparin on in vitro fertilization and subsequent embryonic development in sheep. Small Rumin Res. 63, 226-232. 82 10.1016/j.smallrumres.2005.02.019 Cognie, Y., Baril, G., Poulin, N., Mermillod, P. (2003) Current status of embryo technologies in sheep and goat. Theriogenology 59, 171-188. 83 10.1016/S0093-691X(02)01270-0 Katska-Ksiazkiewicz, L., Opiela, J., Rynska, B. (2007) Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats. Theriogenology 68, 736-744. PMid:17651793 84 10.1016/j.theriogenology.2007.06.016 Izquierdo, D., Villamediana, P., Paramio, M.T. (1999). Effect of culture media on embryo development from prepubertal goat IVM-IVF oocytes. Theriogenology 52, 847-861. 85 10.1016/S0093-691X(99)00177-6 Natarajan, R., Shankar, M.B. Munuswamy, D. (2010). Effect of a-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage. J Assist Reprod Genet. 27, 483-490. PMid:20454845 PMCid:PMC2941590 86 10.1007/s10815-010-9430-7 Natarajan, R., Bhawani, S.M., Munuswamy, D. (2010). Effect of L-ascorbic acid supplementation at different gaseous environments on in vitro development of preimplantation sheep embryos to the blastocyst stage. Anim Reprod. 7, 21-28. 87 Rooke, J.A., McEvoy, T.G., Ashworth, C.J., Robinson, J.J., Wilmut, I., Young, L.E., Sinclair, K.D. (2007). Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction. Theriogenology 67, 639-647. PMid:17070902 88 10.1016/j.theriogenology.2006.09.040 Beilby, K.H., de Graaf, S.P., Evans, G., Maxwell, W.M.C., Wilkening, S., Wrenzycki, C., Grupen, C.G. (2011). Quantitative mRNA expression in ovine blastocysts produced from X- and Y-chromosome bearing sperm, both in vitro and in vivo. Theriogenology 76, 471-481. PMid:21497386 89 10.1016/j.theriogenology.2011.02.024 Thomas, F., Armstrong, D., Telfer, E. (2003). Activin promotes oocyte development in ovine preantral follicles in vitro. Reprod Biol Endocrinol. 1, 76. PMid:14613548 PMCid:PMC280721 90 10.1186/1477-7827-1-76 Hammami, S., Izquierdo, D., Catala, M.G., Paramio, M.T., Morato, R. (2014). In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A. Animal 8, 94-101. PMid:24176127 91 10.1017/S1751731113001936 Thompson, J.G. (2000). In vitro culture and embryo metabolism of cattle and sheep embryos - a decade of achievement. Anim Reprod Sci. 60-61: 263-275. 92 10.1016/S0378-4320(00)00096-8 Lane, M., Gardner, D.K., Hasler, M.J., Hasler, J.F. (2003) Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture. Theriogenology 60, 407-419. 93 10.1016/S0093-691X(03)00030-X Garcia-Garcia, R.M., Ward, F., Fair, S., O'Meara, C.M., Wad, M., Duffy, P., Lonergan, P. (2007). Development and quality of sheep embryos cultured in commercial G1.3/G2.3 sequential media. Anim Reprod Sci. 98, 233-240. PMid:16621355 94 10.1016/j.anireprosci.2006.03.007 Petkov, V., Dovenski, T. (2007). Laparoscopic technique of artificial insemination with frozen semen in small ruminants. 9th Symposium on Veterinary Clinical Pathology and Therapy "Clinica Veterinaria", June, 18-22, Palic, Serbia. 95 Klinc, P., Rath,D. (2006). Application of flowcytometrically sexed spermatozoa in different farm animal species: a review. Arch Anim Breed. 49(1): 41-54. 96 10.5194/aab-49-41-2006 Hollinshead, F.K., O'Brien, J.K., Maxwell, W.M.C., Evans, G. (2002). Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted X- or Y-chromosome-bearing spermatozoa. Reprod Fertil Dev. 14(8): 503. PMid:12617795 97 10.1071/RD02034 De Graaf, S.P., Evans, G., Maxwell, W.M.C., O'Brien, J.K. (2006). In vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex sorting and re-freezing. Reprod Fertil Dev. 18(8): 867. PMid:17147935 98 10.1071/RD06061 Beilby, K.H., Grupen, C.G., Thomson, P.C., Maxwell, W.M.C., Evans, G. (2009). The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram. Theriogenology 71(5): 829-835. PMid:19110307 99 10.1016/j.theriogenology.2008.10.005