Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 127
P300
Acrosin activity evaluation in chilled/rewarmed dog
sperm during different capacitation times
De Los Reyes, M*, Godoy, N; Palomino, J
Animal Production, Animal Reproduction Unit, Faculty of Veterinary Sciences,
University of Chile, Chile
Introduction The acrosome reaction causes release of acrosin, a
proteolytic enzyme that facilitates sperm penetration through the zona
pellucida. The release of acrosin on in vitro capacitated
chilled/rewarmed canine spermatozoa is a time depending process.
Thus, the aim of the present work was to study the proteolytic acrosin
activity in chilled/rewarmed canine spermatozoa during in vitro
capacitation and acrosome reaction.
Methods Six ejaculates were collected from four adult dogs. Each
ejaculate was liquated into 2 fractions and centrifuged in tris buffer
medium. The pellet of one fraction was diluted in fert-talp medium
(fresh control sample), and the pellet of the other fraction was diluted
in a tris-fructose-citric acid and egg-yolk extender and then cooled at
4º C for 24 h. Sperm samples (fresh and chilled/rewarmed) were
centrifuged and the pellet rediluted in fert-talp medium and then
aliquoted into four tubes which were incubated separately for 0, 1, 2
and 3 h at 20º. At each time of cultured, acrosin activity was measured
by the gelatin-substrate film technique, where enzyme activity is
detected by the presence of halos around single sperm resulting from a
localized proteolytic digestion of gelatin. Gelatin suspension was
placed on pre-cooled slides and fixed in 0.05% glutaraldeyde, washed
and kept overnight. Sperm suspensions were placed on the slices and
incubated at 38°C in CO2 for 24 h. Slices were stained with Comassie
Blue and examined with light microscopy for evidence of digestion.
Results Fresh and chilled dog spermatozoa incubated for up to 3 h in
fert-talp medium, displayed digestion halos on gelatin films. Those
digestion halos were of three different mean sizes: small, medium and
large. Acrosin activity as measured by halo diameter on slides coated
with gelatin, showed a significant difference between fresh and
chilled sperm. A low proportion of large halos was observed in fresh
samples at the beginning of culture (time 0 and 1), while chilled
sperm showed a high rate of large halos at these times. During the
following hours (2 and 3) of culture, fresh sperm showed higher rates
of large halos than fresh sperm.
Conclusion These results indicate that acrosomal proteolytic activity
of chilled/ warmed dog sperm is different throughout the time from
that of the control fresh sperm Supported by Grant FONDECYT
1060602.
P301
Terminating pregnancy in bitches: compartive study of
side effects between four treatments
Fila Varela, D 1 *, Berglavaz, A 2 , de Leon, J 3 , Navarro, G 3 , Morgades, D 2 ,
Pereira, O 3 , Elhordoy, D 1 , Cavestany, D 1
1Animal Reproduction Department, Veterinary Faculty, Uruguay; 2 Private
Practitioner, Don Quijote Veterinary, Uruguay; 3 Private Practitioner, Zoolymar
Vet, Uruguay
To compare incidence of side effects in four medical treatments to
terminate unwanted pregnancies. 166 bitches were assigned to
different treatments: Group A: (n=38) aglepristone (Alizine, Virbac,
France) (10 mg/kg), two s.c. injections, 24 hours apart, around 25 to
30 days of gestation; Group D: (n=44) dexamethasone orally (0,2
mg/kg two times per day, by 5 days and then 0,1 mg/kg two times per
day, during 5 more days) around 28 to 30 days of gestation; Group E:
(n=45) estradiol (0,01mg/kg) intramuscularly within 48 hours of
mated; and Group L: (n=39) lotrifen (Privaprol, FATRO, Italy) (2,5
mg/kg) intramuscularly within 15 days of mated. In group A, there
were no side effects. In group D, the side effects were mild polydipsia
and polyuria that disappeared when treatment was discontinued.
Moreover, 7 of 44 bitches (15,91%) presented bloody vaginal
discharge during 7 days after treatment ended, without general
symptoms. In group E, 12 of 45 bitches (26,67%) developed cystic
endometrial hyperplasia-pyometra that forced ovary-hysterectomy. In
group L, 18 of 39 bitches (46,15%) developed cystic endometrial
hyperplasia-pyometra that forced ovary-hysterectomy. Data were
analyzed using X2 test and differences within treatments were
considered significant when p 0.05). They occurred in 6/6, 3/3,
5/6 and 5/6, 2/3, 4/5 of the animals of the DA, DA&ACY and
DA&ACY+2 groups, respectively. Estrous response appeared 5.0 ±
1.2, 10 ± 1.0 and 24.2 ± 11.7 days after treatment in the same groups
(P = 0.1).
Conclusions The GnRH antagonist, acyline, administered at two
different time points after a GnRH agonist failed to prevent estrous
response in most of these anestrous bitches. Although, in the
antagonist treated groups, estrous response had a tendency to appear
later. These findings suggest a postponement of the stimulation period
during the peak antagonistic effect. Further studies with repeated or
higher doses of antagonists are necessary.
Acknowledgements This study was funded by the National Agency
for Scientific and Technological Promotion, Argentina (Grant PICT
38376/05). The authors thank to NICHHD, NIH, USA and Peptech,
Australia for drug provision.
References Wright PJ, et al. 2001. The Suppression by Progestin of
Oestrus Responses of the Bitch to the GnRH Analogue Deslorelin. J
Reprod Fertil. 57: 283-8.
P303
Distribution of active mitochondria in canine oocytes is
related to reproductive cycle stage but can be damaged
during IVM culture
Iorga, AI*; Valentini, L; De Santis, T; Ambruosi, B; Guaricci, AC; Caira, M;
Dell’Aquila, ME
Department of Animal Production, University of Bari, Italy
Introduction We investigated the effect of the reproductive cycle
stage on the distribution of active mitochondria in canine oocytes
examined 1) at collection and 2) after in vitro maturation (IVM).
Methods Cumulus-oocyte complexes (ooplasmic size >120 µm in
diameter) were recovered from 20 bitches divided into five groups