Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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