Developing Assisted Reproductive Technologies in Banteng (Bos javanicus) and Lowland Anoa (Bubalus depressicornis) James Gillis MS, Linda Penfold PhD Why? Logistics of animal transport Genetic compatibility ≠ behavioral compatibility Reduction of stress Dissemination of genetics & genome resource banking Provides tools for the GSMP Anesthesia Protocol for Semen Collection Drug Dose Butorphanol 0.083 mg/kg Azaperone 0.027 mg/kg Medetomidine 0.034 mg/kg http://wildpharm.com/medications/wildlife/item/61-bamkit.html Semen Collection by Electroejaculation Treatment Volume Concentration Total Sperm Count Motility Status Morph. pH Osmolarity (ml) ( 106/ml) ( 106) (%) (1-5) Normal (mOsm) (%) With Oxytocin 22 ± 7.5 2380 ± 858.8 32275 ± 8813.7 81 ± 5 3 ± 0.1 92 ± 2.8 7 ± 0.2 301 ± 3.8 (n=6) Without 9.8 ± 1.4 365 ± 77.6 3683 ± 1006.5 81 ± 2.4 3 ± 0.1 81 ± 2.4 7 ± 0..2 299 ± 2.3 Oxytocin (n=10) Avg. 14 ± 3.2 1120 ± 397 12205 ± 4781 81 ± 2.2 3 ± 0.1 88 ± 3 7.1 ± 0.1 300 ± 2 Oxytocin Increases Sperm Yield Total Sperm Count in Males Treated With & Without Oxytocin 60000 50000 6 0 40000 1 × m r e 30000 p Males W/O Oxytocin s l a t Males W/ Oxytocin o T 20000 10000 0 0 2 4 6 8 10 12 Age (years) Cold Storage & Cryopreservation of Sperm Methods adopted from domestic bull protocols Banteng sperm maintains motility in both egg-yolk and whole milk based extenders for 24 h at 5°C Sperm frozen in an egg-yolk Tris diluent (Biladyl) with 5-7% glycerol provided the highest post-thaw motility of 43% compared to TEST and whole milk diluents at 26% and 29%, respectively Estrus Synchronization for Artificial Insemination 2× Lutalyse Injections CO-Synch CIDR removal in a banteng Ovsynch Banteng Artificial Insemination The cervix in banteng is more caudal than in domestic cattle. A plastic pliable catheter is used for semen deposition, to mitigate the risk of trauma in a restrained, non- anesthetized animal. Defining Success Institution Number of AI Pregnancy Rate Offspring Attempts Production St. Louis Zoo 2 2 0 Western Plains 6 1 1 Zoo The Wilds 9 1 1 CCTU & SEZARC 4 0 0 *CCTU & SEZARC* 12 0 0 CCTU & SEZARC 22 1 1 Indonesia ? 1 1 Bali Cattle - 70-99% 70-99% (domestic) Serum Progesterone Profile for Banteng 45 CCTU White Oak 40 35 ) l m 30 / g n ( e 25 n o r e t 20 s e g o 15 r P 10 5 0 Is the Synchronization 7 8 0 1 3 4 6 8 0 5 6 2 3 4 8 9 0 1 2 3 5 1 0 9 5 5 8 1 1 2 2 Y Y Y Y 1 1 1 2 2 2 2 2 3 3 33 33 5 4 35 1 1 81 5 3 4 R R R R R R Y Y Y Y Y Y Y Y Y Y 2 7 0 0 9 0 0 9 0 0 0 8 8 0 0 8 0 0 8 0 0 0 95 93 94 94 93 94 95 Avg = 8.38 ng/ml Animal ID Avg = 14.33 ng/ml Working? Serum Cortisol Profile for Banteng 200 CCTU White Oak 180 160 140 ) l m 120 / g n ( l 100 o s i t r 80 o C 60 40 20 0 7 8 0 1 3 4 6 8 ) ) 3 3 5 1 5 8 1 1 2 2 Y Y Y Y 10 15 16 22 23 24 28 29 30 31 e le 3 3 2 3 3 5 1 8 0 9 5 R R R R R R Y Y Y Y Y Y Y Y Y Y al a 2 7 5 4 9 1 1 9 5 3 4 8 8 00 00 8 00 00 8 00 00 00 M M 5 3 4 4 3 4 5 ( ( 9 9 9 9 9 9 9 Y4 2 04 Y Avg (Female)= 102.26 ng/ml AnimaStressl ID and Success?Avg = 39.82 ng/ml Objectives Objective 1. Effects of oxytocin on semen volume and sperm concentration. Objective 2. Comparison of varying extenders and temperature on short- term sperm storage. Objective 3. Effects of cryoprotectant concentrations and cholesterol- loaded cyclodextrins on sperm motility and on sperm cryopreservation. Objective 4. Fertilization rates of cholesterol-loaded cyclodextrin treated frozen-thawed sperm using heterologous in-vitro fertilization. Objective 5. Characterization of lowland anoa estrous cycle through non- invasive fecal hormone and behavioral monitoring. Objective 6. Effects of exogenous prostaglandins on lowland anoa corpus luteum and efficacy in manipulation of the estrous cycle for artificial insemination. .