Proceedings Annual Conference of the International Embryo Transfer Society, Nice Acropolis, Nice, France, Jan., 1997

BRACKETT, B. G.

KESKINTEPE, L.

SIMPLICIO, A. A.

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Theriogenology 299 USE OF CRYOPRESERVED SPERMATOZOA FOR CAPRINE IN VITRO FERTILIZATION (IVF) A.A.Simplicio 192 , B.G.Brackett’, and L.Keskintepe’ 1 Department of Physiology & Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602; 2Brazilian Agricultural Research Agency National Goat Research Center, PO Box D-10, Sobral, Ceara, Brazil. Feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Two Nubian and 1 Nubian-Boer bucks were electroejaculated. Sperm were washed 2x (Proc.Rec.Adv. in Goat Prod. 2:1089-1094), frozen in skim milk (Brazilian Soc.Anim.Reprod. 171-177), or in egg-yolk (World RevAnim. Prod. 8:80). Oocytes with 2-4 layers of cumulus cells from 3-6 mm follicles washed in Tyrode’s with pyruvate and PVA, were incubated in 1 ml of Hepes-TCM-199 + 10 ug oFSH and 10 pg bLH (NHPP, NIDDK, NICHD, USDA)/ml + 20% FBS (MM) at 38.5’C. After 4.5 h oocytes were further incubated (23 h) in 75 ul of MM under paraffin oil and 5% 02, 5% CO*, 90% NZ. Frozen sperm were thawed in a water bath (37°C 15 set), and selected by swim-up. IVF was in mDM (Theriogenology 37: 1049-1060), supplemented as below, for 24 h. Embryo culture was in 50 ul of c-SOF+NEA (Biol. Reprod. 55:333-339) for 9 d. Data analysis was by ANOVA and Bonferroni t-test. Percentages of oocytes exposed to heparin-capacitated (HC) sperm that reached cleavage (C), morula (M), blastocyst (B), and expanded B (EB) were 82.8, 57.1, 35.7, 30.0 %, respectively; without heparin treatment of sperm data for C, M, B, EB were 44.3, 3 1.4, 18.6, 8.6 %, respectively. Further work employed HC sperm. Use of cryopreserved sperm with BSA for IVF yielded no C. Although extenders containing 8 to 20 % egg-yolk enabled good motility after crypreservation in vitro fertilizing ability was lost in our conditions. By contrast, commercial semen processed in season with egg-yolk was effective for IVF (see table). Table. Caprine IVF with Cryopreserved Spermatozoa Development stages / Inseminated oocytes (%) Freezing Supplof No. of Cleaved Morula Blastocyst Expanded extender IVF oocytes 48 h 120h (B) B medium inseminated 168 h 216 h Skim Milk LS’ 40 33 (82.5)a 20 (5O.O)c 12 (30.0)n 9 (22.5)’ Skim Milk FCS 37 4 (10.8)b 1 (2.7)e Oi Orn Egg Yolk C2 Ls 41 24 (58.5)’ 11 (26.8)’ 7 (17.1)j 4 (9.7)” Egg Yolk C FCS 38 6 (15.8)b 4 ( lo.5)g 3 (7.9)k Orn ‘Lamb serum. ‘Sperm processed in season in egg yolk commercially. “D ifferent superscripts in the same column denote significant differences (PcO.05). Highest proportions of blastocysts resulted after sperm cryopreservation i skim milk extender, heparin capacitation, and insemination in media containing lamb serum. 

Use of cryopreserved spermatozoa for caprine in vitro fertilization (IVF).

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