Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
174 Poster Abstracts<br />
P444<br />
Effective low dose <strong>in</strong>sem<strong>in</strong>ation with sex-sorted ram<br />
spermatozoa<br />
Beilby, K*; Grupen, C; Maxwell, WMC; Evans, G<br />
The Faculty of Veter<strong>in</strong>ary Science, The University of Sydney, Sydney,<br />
Australia<br />
Sex-sorted ram sperm is an attractive breed<strong>in</strong>g management tool <strong>in</strong><br />
commercial production systems. However, the time required and cost<br />
associated with sort<strong>in</strong>g large commercial sperm doses limits the use of<br />
this technology. Previous research has shown that <strong>in</strong>sem<strong>in</strong>ation with<br />
sex-sorted ram sperm resulted <strong>in</strong> equal, if not superior, fertility<br />
compared to non-sorted controls at dose rates of 1, 5 and 15 million<br />
motile sperm 1 . Pregnancy rates were similar for sex-sorted sperm at<br />
these dose rates. Therefore, the current study aimed to <strong>de</strong>term<strong>in</strong>e the<br />
m<strong>in</strong>imum dose that could be <strong>in</strong>sem<strong>in</strong>ated before fertility was<br />
significantly reduced. Semen was collected from 3 mer<strong>in</strong>o rams. Half<br />
of each ejaculate was frozen immediately (non-sorted, control sperm)<br />
and the rema<strong>in</strong><strong>in</strong>g half was sex-sorted via modified flow cytometry<br />
and then frozen (sorted sperm). Oestrus was synchronised <strong>in</strong> ewes<br />
(N=138) us<strong>in</strong>g progestagen pessaries (12 days) and PMSG at pessary<br />
removal (PR). Ovulation was further controlled us<strong>in</strong>g GnRH given<br />
36h after PR. All animals were <strong>in</strong>sem<strong>in</strong>ated by laparoscopy 58h after<br />
PR with either 5 x 10 5 (n = 60), 1 x 10 6 (n = 39) or 15 x 10 6 (n = 39)<br />
sorted or non-sorted, frozen-thawed motile spermatozoa. Pregnancy<br />
rates were assessed at day 60 after <strong>in</strong>sem<strong>in</strong>ation us<strong>in</strong>g cutaneous realtime<br />
ultrasound. There was no difference <strong>in</strong> pregnancy rate for groups<br />
<strong>in</strong>sem<strong>in</strong>ated with sorted or non-sorted spermatozoa across all dose<br />
rates. Furthermore, there was no difference between doses of 1 and 15<br />
million spermatozoa. However, the pregnancy rates were lower (P <<br />
0.05) for the 5 x 10 5 sperm dose (15 and 13%, for non-sorted and<br />
sorted, respectively) compared with doses of 1 (49 and 41%) and 15 x<br />
10 6 spermatozoa (44 and 49%). More than 90% of lambs born were of<br />
the predicted sex. This study confirmed that a dose of 1 x 10 6 sexsorted<br />
frozen-thawed motile sperm is the m<strong>in</strong>imum that can be used<br />
for laparoscopic <strong>in</strong>sem<strong>in</strong>ation of ewes before fertility is reduced. This<br />
is much lower than the current <strong>in</strong>dustry standard of 20-40 x 10 6<br />
frozen-thawed sperm/<strong>in</strong>sem<strong>in</strong>ate. These results provi<strong>de</strong> the sheep<br />
<strong>in</strong>dustry with a promis<strong>in</strong>g assisted reproductive management strategy<br />
that is not only effective, but practically applicable.<br />
P445<br />
Advantages of the association of glutam<strong>in</strong>e and LDL (Low<br />
Density Lipoprote<strong>in</strong>) for freez<strong>in</strong>g can<strong>in</strong>e sperm<br />
Bencharif, D 1 *, Ta<strong>in</strong>turier, D 1 , Pascal, O 1 , Larrat, M 1 , Langlois, ML 2 , Barrière,<br />
JP 2 , Amirat-Briand, L 1<br />
1Department of Biotechnologies and Reproductive Pathology, Nantes<br />
Veter<strong>in</strong>ary College, Nantes, France; 2 Department of Reproductive Pathology,<br />
Mother and Child, CHU Hôtel Dieux, Nantes, France<br />
Introduction Various studies have <strong>de</strong>monstrated the cryoprotective<br />
action of glutam<strong>in</strong>e (Glut). The latter improves spermatozoa motility<br />
and provi<strong>de</strong>s superior protection for the cytoplasmic membrane. This<br />
prelim<strong>in</strong>ary study aims to <strong>de</strong>term<strong>in</strong>e the i<strong>de</strong>al concentration of<br />
glutam<strong>in</strong>e when comb<strong>in</strong>ed with 6% LDL to improve the<br />
cryopreservation of can<strong>in</strong>e semen.<br />
Materials and method Exp n°1: 20 ejaculates were collected from 6<br />
dogs (Beagle, Gol<strong>de</strong>n Retriever) aged from 3 to 6 years. Semen with a<br />
motility of between 2 and 5 were frozen <strong>in</strong> different media: Basic<br />
Medium (BM)+20% egg yolk (e.y), BM+6% LDL, and BM+6%<br />
LDL+60, 70, 80, 90, or 100 mmol of Glut. Follow<strong>in</strong>g collection, the<br />
spermatic and prostatic fractions were mixed and diluted <strong>in</strong> the<br />
different freez<strong>in</strong>g media at +37°C to obta<strong>in</strong> a f<strong>in</strong>al concentration of<br />
100x10 6 spermatozoa/ml. Exp n°2: In the same way as for Exp n°1,<br />
the ejaculates were frozen <strong>in</strong> different media: BM+20% e.y, BM+6%<br />
LDL, and BM+6% LDL+10, 20, 30, 40, or 50 mmol of Glut. Exp n°3:<br />
10 ejaculates were collected from these same 5 dogs, and frozen <strong>in</strong>:<br />
BM+20% e.y, 6% LDL milieu (e.y extract), and 6 % LDL<br />
medium+20 mmol Glut. The semen was cooled to +4°C for 1 hour<br />
then aspirated <strong>in</strong>to 0.25 ml straws and stored for 30 m<strong>in</strong> at +4°C. The<br />
straws were placed <strong>in</strong> the liquid nitrogen vapours at -110°C for 10<br />
m<strong>in</strong>utes, before be<strong>in</strong>g plunged <strong>in</strong>to the liquid nitrogen. The straws<br />
were thawed <strong>in</strong> a water bath at +37°C for 30 seconds. The semen was<br />
assessed 10 m<strong>in</strong>utes after thaw<strong>in</strong>g with a HAMILTON THORN<br />
CERROS 12 image analyser. For Exp 3, spermatozoa <strong>in</strong>tegrity was<br />
analysed us<strong>in</strong>g Acrid<strong>in</strong>e orange, HOS, PSA-FITC, and Spermac ®<br />
tests.<br />
Results Exp n°1: the motility results were superior <strong>in</strong> the 6% LDL<br />
media than the other media (BM+20% (e.y), BM+6% LDL+60, 70,<br />
80, 90, 100 mmol of Glut) (43,13% vs 27,3, 28,7, 30,9, 24,1, 31,1,<br />
27,8% respectively) (p