Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 174 Poster Abstracts P444 Effective low dose insemination with sex-sorted ram spermatozoa Beilby, K*; Grupen, C; Maxwell, WMC; Evans, G The Faculty of Veterinary Science, The University of Sydney, Sydney, Australia Sex-sorted ram sperm is an attractive breeding management tool in commercial production systems. However, the time required and cost associated with sorting large commercial sperm doses limits the use of this technology. Previous research has shown that insemination with sex-sorted ram sperm resulted in equal, if not superior, fertility compared to non-sorted controls at dose rates of 1, 5 and 15 million motile sperm 1 . Pregnancy rates were similar for sex-sorted sperm at these dose rates. Therefore, the current study aimed to determine the minimum dose that could be inseminated before fertility was significantly reduced. Semen was collected from 3 merino rams. Half of each ejaculate was frozen immediately (non-sorted, control sperm) and the remaining half was sex-sorted via modified flow cytometry and then frozen (sorted sperm). Oestrus was synchronised in ewes (N=138) using progestagen pessaries (12 days) and PMSG at pessary removal (PR). Ovulation was further controlled using GnRH given 36h after PR. All animals were inseminated by laparoscopy 58h after PR with either 5 x 10 5 (n = 60), 1 x 10 6 (n = 39) or 15 x 10 6 (n = 39) sorted or non-sorted, frozen-thawed motile spermatozoa. Pregnancy rates were assessed at day 60 after insemination using cutaneous realtime ultrasound. There was no difference in pregnancy rate for groups inseminated with sorted or non-sorted spermatozoa across all dose rates. Furthermore, there was no difference between doses of 1 and 15 million spermatozoa. However, the pregnancy rates were lower (P < 0.05) for the 5 x 10 5 sperm dose (15 and 13%, for non-sorted and sorted, respectively) compared with doses of 1 (49 and 41%) and 15 x 10 6 spermatozoa (44 and 49%). More than 90% of lambs born were of the predicted sex. This study confirmed that a dose of 1 x 10 6 sexsorted frozen-thawed motile sperm is the minimum that can be used for laparoscopic insemination of ewes before fertility is reduced. This is much lower than the current industry standard of 20-40 x 10 6 frozen-thawed sperm/inseminate. These results provide the sheep industry with a promising assisted reproductive management strategy that is not only effective, but practically applicable. P445 Advantages of the association of glutamine and LDL (Low Density Lipoprotein) for freezing canine sperm Bencharif, D 1 *, Tainturier, D 1 , Pascal, O 1 , Larrat, M 1 , Langlois, ML 2 , Barrière, JP 2 , Amirat-Briand, L 1 1Department of Biotechnologies and Reproductive Pathology, Nantes Veterinary College, Nantes, France; 2 Department of Reproductive Pathology, Mother and Child, CHU Hôtel Dieux, Nantes, France Introduction Various studies have demonstrated the cryoprotective action of glutamine (Glut). The latter improves spermatozoa motility and provides superior protection for the cytoplasmic membrane. This preliminary study aims to determine the ideal concentration of glutamine when combined with 6% LDL to improve the cryopreservation of canine semen. Materials and method Exp n°1: 20 ejaculates were collected from 6 dogs (Beagle, Golden Retriever) aged from 3 to 6 years. Semen with a motility of between 2 and 5 were frozen in different media: Basic Medium (BM)+20% egg yolk (e.y), BM+6% LDL, and BM+6% LDL+60, 70, 80, 90, or 100 mmol of Glut. Following collection, the spermatic and prostatic fractions were mixed and diluted in the different freezing media at +37°C to obtain a final concentration of 100x10 6 spermatozoa/ml. Exp n°2: In the same way as for Exp n°1, the ejaculates were frozen in different media: BM+20% e.y, BM+6% LDL, and BM+6% LDL+10, 20, 30, 40, or 50 mmol of Glut. Exp n°3: 10 ejaculates were collected from these same 5 dogs, and frozen in: BM+20% e.y, 6% LDL milieu (e.y extract), and 6 % LDL medium+20 mmol Glut. The semen was cooled to +4°C for 1 hour then aspirated into 0.25 ml straws and stored for 30 min at +4°C. The straws were placed in the liquid nitrogen vapours at -110°C for 10 minutes, before being plunged into the liquid nitrogen. The straws were thawed in a water bath at +37°C for 30 seconds. The semen was assessed 10 minutes after thawing with a HAMILTON THORN CERROS 12 image analyser. For Exp 3, spermatozoa integrity was analysed using Acridine orange, HOS, PSA-FITC, and Spermac ® tests. Results Exp n°1: the motility results were superior in the 6% LDL media than the other media (BM+20% (e.y), BM+6% LDL+60, 70, 80, 90, 100 mmol of Glut) (43,13% vs 27,3, 28,7, 30,9, 24,1, 31,1, 27,8% respectively) (p
16 t h International Congress on Animal Reproduction Poster Abstracts 175 P447 The effect of plant lipid extender component on quality of frozen ram semen Palasz, A 1 , Bochenek, M 2 *, Gogol, P 2 , Kareta, W 2 , Smorag, Z 2 1INIA, Dpto Reproduccion Animal, Crta. Coruña, km 5, 9-28040 Madrid, Spain; 2 Dept. Biotechnology of Animal Reproduction, NRIAP, 32-083 Balice, Poland Experiment was designed to test different concentrations and different homogenization protocols of soybean lipids liposomes for the use as a milk replacement in ram semen freezing extender. Lipids were homogenized by high pressure homogenization (800 bars) and then 5% (Y1 extender) or 10% (Y2 extender) liposomes were mixed with 8% glycerol. As additional group, lipids and glycerol were homogenized together (LIPO extender). All 3 extenders (Y1, Y2, LIPO) were prepared in Tris buffer containing citric acid and fructose. As control group milk/egg yolk (MY) extender was used. The semen was collected from 3 rams with artificial vagina. Post thaw motility and survival time (total of 11 ejaculates), lipid peroxidation (total of 7 ejaculates) and sperm membrane integrity (total of 3 ejaculates) were examined in 4 extenders tested. Immediately after collection each ejaculate was divided into 4 parts, diluted with Y1, Y2, LIPO and MY extenders and processed according schedule. Post thaw motility and survival time: Mean sperm motility examined immediately after thawing for Y1, Y2, LIPO and MY extenders was 51.4%, 46, 8%, 50.6% and 44.4% respectively. Mean survival time of spermatozoa kept at 42°C was 200, 220, 255 and 135 minutes for Y1, Y2, LIPO and MY extenders respectively. Lipid peroxidation was monitored by chemiluminescence method. Iron induced luminescence of frozen/thawed sperm cells was assessed using a luminometer. It was shown that Y1, Y2 and LIPO semen extenders had significantly lower peroxidation level than MY extender. The values of Integral parameter for Y1, Y2, LIPO, MY extenders was 4,57; 5,49; 4,31; 28,70 respectively. Sperm membrane integrity: After thawing semen sample was diluted with PBS to 20mln/ml concentration and kept for 1h at room temperature. Sperm membrane examination (“live/dead”) was performed by double staining with SYBR-14/propidium iodide fluorochromes and analysis by flow cytometry. Data of 20 000 spermatozoa were collected for each sample. The percentage of membrane intact (“live”) spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa for Y1, Y2, lipo and milk extenders were 10.79%, 10.61%, 10.82% and 4.71% respectively. Statistically significant differences was found between milk and Y1, Y2, LIPO (test t, P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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