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 />
Poster Abstracts 125<br />
only to the ipsilateral ovary but are transported with<strong>in</strong> the<br />
mesometrium to both ovaries <strong>in</strong> a more systemic manner.<br />
Poster 07 - <strong>Reproduction</strong> of Pet Carnivores<br />
P294<br />
IVF of <strong>in</strong> vitro matured dog oocytes us<strong>in</strong>g homologous<br />
fresh or frozen-thawed spermatozoa<br />
Alhai<strong>de</strong>r, AK 1 *; Satake, N 1, 2 ; Watson, PF 1<br />
1Veter<strong>in</strong>ary Basic Sciences, Royal Veter<strong>in</strong>ary College, UK; 2 Institute of<br />
Zoology, Zoological Society of London, UK<br />
Fertilization is the crucial test of both successful oocyte maturation<br />
and sperm cryopreservation. The aims of this study were to<br />
<strong>in</strong>vestigate the effect of sperm freez<strong>in</strong>g and the <strong>in</strong>fluence of sperm<br />
donor on the outcome of IVF of oocytes cultured un<strong>de</strong>r conditions<br />
<strong>de</strong>veloped <strong>in</strong> our laboratory and to <strong>in</strong>vestigate the effect of Percollwash<br />
frozen-thawed spermatozoa on IVF outcome. Cumulus oocyte<br />
complexes (COCs) were collected from spay ovaries and cultured <strong>in</strong><br />
TCM 199 medium supplemented with 0.3 % BSA, 7 μg/ml<br />
progesterone, 100 nM each of growth hormone, IGF-I, FGF and TGFα<br />
for 48 h at 39 °C <strong>in</strong> 5 % CO2 <strong>in</strong> air <strong>in</strong> a humidified <strong>in</strong>cubator. COCs<br />
were then co-<strong>in</strong>cubated with 1 x 10 6 /ml capacitated-fresh or frozenthawed<br />
spermatozoa from the same dog for 12 h. COCs were cultured<br />
for further 48 h before be<strong>in</strong>g <strong>de</strong>nu<strong>de</strong>d and fixed. Oocytes were sta<strong>in</strong>ed<br />
with propidium iodi<strong>de</strong> and then evaluated un<strong>de</strong>r a laser scann<strong>in</strong>g<br />
confocal microscope. Fertilization rate was significantly higher <strong>in</strong><br />
oocytes <strong>in</strong>sem<strong>in</strong>ated with fresh semen (55.8 vs 37.2 %, P = 0.001).<br />
There were no significant differences between oocytes fertilized with<br />
fresh and frozen-thawed semen <strong>in</strong> cleavage rate (26.7 vs 18.5 %<br />
respectively) and polyspermic fertilization (41.9 vs 50.0 %<br />
respectively). However, significantly more embryos at the 2-cell stage<br />
were recor<strong>de</strong>d when fresh spermatozoa were used for fertilization (P<br />
< 0.01). Individual analysis of each dog ejaculate revealed differences<br />
<strong>in</strong> their sperm fertiliz<strong>in</strong>g ability after freez<strong>in</strong>g. Freeze-thaw<strong>in</strong>g<br />
significantly <strong>de</strong>creased fertilization rate <strong>in</strong> Dogs 2 and 4 but had no<br />
effect on spermatozoa from Dogs 1 and 3. When the same analysis<br />
was employed for cleavage rate, freeze-thaw<strong>in</strong>g significantly<br />
<strong>de</strong>creased the number of cleaved embryos <strong>de</strong>rived from COCs<br />
fertilized with semen from Dog 1 only (fresh: 33.3, frozen-thawed:<br />
8.0 %, P = 0.025). Percoll-wash had no effect on fertilization and<br />
cleavage rates. In conclusion, freez<strong>in</strong>g-thaw<strong>in</strong>g significantly reduced<br />
fertilization rate <strong>in</strong> vitro, but not cleavage rate. Sperm donors<br />
<strong>in</strong>fluenced fertilization and cleavage rates <strong>in</strong> both fresh and frozenthawed<br />
semen. Moreover, oocytes matured <strong>in</strong> vitro un<strong>de</strong>r the<br />
conditions <strong>de</strong>veloped <strong>in</strong> our laboratory were found to be competent to<br />
achieve fertilization and susta<strong>in</strong> early embryonic <strong>de</strong>velopment up to<br />
48 h of IVC.<br />
P295<br />
Centrifugation and dilution effects on sperm quality and<br />
freezability from dog<br />
Nicolas, M 1 , Mata-Campuzano, M 1 , Gomes-Alves, S 2 , Garcia-Macias, V 1 ,<br />
Alvarez, M 1 , Tamayo, J 1 , De Paz, P 2 , Anel, L 1 *<br />
1Animal <strong>Reproduction</strong> and Obstetrics, University of Leon, Spa<strong>in</strong>; 2 Cell<br />
Biology, University of Leon, Spa<strong>in</strong><br />
Semen process<strong>in</strong>g before cryopreservation <strong>in</strong> wild animals<br />
(electroejaculated) <strong>in</strong>volves centrifugation. Dog ejaculates could be an<br />
appropriate animal mo<strong>de</strong>l to study semen manipulation <strong>in</strong> endangered<br />
wild carnivores, <strong>in</strong> our case cantabric brown bear (Ursus arctos). The<br />
aim of this study was to assess the effects of semen dilution before<br />
centrifugation at 4 different dilution rates <strong>in</strong> dog spermatozoa. Sperm<br />
rich fractions of ejaculates from 8 healthy dogs (means concentration<br />
505.72x106 spermatozoa/ml), collected by digital manipulation, were<br />
divi<strong>de</strong>d <strong>in</strong>to 5 aliquots. 4 were diluted (Tris, glucose, citric acid,<br />
antibiotics) at: 1:1 (a), 1:4 (b), 1:8 (c) and 1:16 (d) dilution rates, and<br />
the other was not diluted (control) (e). All the aliquots were<br />
centrifuged at 600 g for 6 m<strong>in</strong>utes (Centrifuge 2-15, Sigma), then<br />
supernatant was immediately removed. Each pellet was suspen<strong>de</strong>d<br />
us<strong>in</strong>g a freez<strong>in</strong>g exten<strong>de</strong>r (centrifugation exten<strong>de</strong>r with 7% glycerol<br />
and 20% egg yolk) to a f<strong>in</strong>al concentration of 100x106<br />
spermatozoa/ml, cooled at -0.25ºC/m<strong>in</strong> and equilibrated at 5ºC for 1<br />
hour. Diluted semen was placed <strong>in</strong>to 0.25 ml straws, sealed (Ultraseal<br />
21, M<strong>in</strong>itub) and frozen from 5ºC to -100ºC (-20ºC/m<strong>in</strong>) <strong>in</strong> a<br />
programmable cell freezer (Kryo 10, Planer). Straws were plunged<br />
<strong>in</strong>to liquid nitrogen until analysis and thawed <strong>in</strong> a water bath (65ºC 6<br />
s). Semen quality was assessed at 3 different stages: immediately after<br />
the collection, postcentrifugation and after thaw<strong>in</strong>g. Motility (total<br />
motility TM, %; progressive motility PM, %) was assessed with<br />
computer assisted sperm analyzer (SCA, Microptic, Barcelona).<br />
Viability (%) was assessed by flow citometry us<strong>in</strong>g SYBR-14 and<br />
propidium iodi<strong>de</strong> sta<strong>in</strong><strong>in</strong>g. Motility parameters <strong>de</strong>creased accord<strong>in</strong>g to<br />
the <strong>in</strong>crease of the dilution rate. Total and progressive motility had<br />
lower values <strong>in</strong> postcentrifugate (TM: a 72.92; b 74.04; c 71.33; d<br />
61.72; e 72.19; PM: a 29.16; b 24.82; c 17.07; d 16.18; e 29.83) and<br />
after thaw<strong>in</strong>g samples (TM: a 32.98; b 41.51; c 44.52; d 48.86; e<br />
40.70; PM: a 17.71; b 22.41; c 25.53; d 22.53; e 21.79) than <strong>in</strong> fresh<br />
semen (TM:86.77; MP:50.23). Thus, total motility was lower <strong>in</strong><br />
postcentrifugate 1:16 diluted samples than <strong>in</strong> fresh semen show<strong>in</strong>g<br />
statistical differences (p0.05).<br />
In conclusion, cat semen exten<strong>de</strong>d and frozen with EY-TFC and EY-<br />
SMGT exten<strong>de</strong>rs, have been successful <strong>in</strong> post thaw motility and<br />
morphological <strong>de</strong>fect rates. However, EED and aborts was thought to