Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
Poster Abstracts 189
P490
Use of immunomodulation in susceptible mares to clear
an experimentally induced endometritis effect on proinflammatory
cytokines: IL-1β, IL-6 and IL-8 mRNA
expression
Fumuso, E* 1 ; Giguère, S 2 ; Rogan, D 4 , Rivulgo, V 1 ; Wade, J 3 ; Rodriguez, E 1 ;
and Sánchez Bruni S 1
1Faculty of Veterinary Medicine, UNCPBA, Tandil, Argentina 7000, Argentina;
2Coll.Vet. Med. Gainesville, FL, USA; 3 Ireland; 4 Bioniche Life Sciences Inc,
Belleville, Ontario, Canada
The effect of an immunomodulator, Mycobacterial Cell Wall Extract
(MCWE), on IL-1β; IL-6 and IL-8 mRNA expression was studied in
mares susceptible to endometritis after experimental uterine infection
with Streptococcus zooepidemicus. Thirty endometritis susceptible
mares, based on the presence of uterine fluid during both diestrus and
estrus, were inoculated with 5 × 10 6 CFU of S. zooepidemicus on day
1 of estrus. Twenty-four hours later, the progression of infection was
evaluated by ultrasonography, bacteriology, exfoliative cytology, and
uterine biopsy. Forty eight hours after inoculation and confirmation of
uterine infection, mares were randomly assigned to one of four
unbalanced experimental groups in order to receive: Group A: 1500
μg total dose of MCWE (Settle) by intrauterine route (IU) (n = 10),
Group B: the same treatment as Group A, but using the intravenous
(IV) route (n = 10), Group C: Distilled water as placebo (PL) IU (n =
5) and the Group D: was treated as Group C using the IV route (n =
5). Endometrial biopsies were taken when infection was confirmed
(day 0), at ovulation (day 3) and 7 days post-ovulation (day 10) for
measurement IL-1β; IL-6 and IL-8 mRNA expression. Total RNA
was isolated, treated with DNAse-I and cDNA was synthesized.
Relative quantitation of mRNA expression (RmRNA) was determined
by real-time PCR. The effect of treatment (MCWE vs PL),
administration route (IU vs IV), and day of sampling (0, 3, 10) on
RmRNA mixed model analysis of a split-unit experiment with
repeated observations. There was no effect of route of administration
on RmRNA. As a result Groups A & B (treated) and groups C & D
(PL), were combined in the final analysis. There were no significant
differences in RmRNA between PL and treated groups on day 0. On
days 3 and 10, MCWE treated mares had significantly lower IL-1β,
IL-6 and IL-8 mRNA expression than mares in the PL group. We
previously demonstrated the effect of immunomodulation after AI in
susceptible mares to post breeding endometritis (Fumuso et al. 2003,
2007); this effect was confirmed in the present study through
experimentally induced endometritis. Results indicate that MCWE
exerts an anti-inflammatory effect on cytokine RmRNA that may
contribute to resolution of endometritis caused by S. zooepidemicus in
mares.
P491
The addition of malondialdehyde during in vitro
maturation of bovine oocytes improves subsequent
cleavage rate after in vitro fertilization
Garcia-Ispierto, I 1 *, Leroy, JLMR 2 ; Lopez-Béjar, M 1 ; López-Gatius, F 3 ; De
Clercq, JPB 2 ; Andries, S 2 ; Goovaerts, IGF 2 ; Bols, PEJ 2
1Department of Animal Health and Anatomy, Autonomous University of
Barcelona, Spain; 2 Laboratory of Veterinary Physiology, Department of
Veterinary Sciences, University of Antwerp, Belgium; 3 Department of Animal
Production, University of Lleida, Spain
Introduction Oxidative stress has been related to heat shock on
embryos and has been considered to play a critical role in the success
of in vitro fertilization protocols. Malondialdehyde (MDA) is the
major endogenous product of lipid peroxidation due to oxidative
stress. It has been demonstrated that MDA can have toxic effects on
bacterial and mammalian cells. Oxidative stress is high during the
postpartum period and this can be exacerbated by heat stress during
the hot seasons.
Objective The aim of the current study was to apply MDA combined
with high temperature conditions during in vitro maturation of oocytes
to evaluate possible effects on oocyte developmental competence.
Methods Bovine ovaries were obtained from the slaughterhouse. A
total of 1209 immature Grade I cumulus–oocyte complexes (COCs)
were aspirated from follicles 2–6mm of diameter. COCs were
cultured in groups of 50 for 24 h in 500µl serum-free maturation
medium (20 ng/ml mEGF) with or without MDA (8µM) under normal
(38.5ºC) or high temperature (41ºC) conditions in a humidified 5%
CO 2 incubator. Four treatment groups were used during maturation:
control (C), MDA (M), high temperature (T) and MDA plus high
temperature (TM). After IVM, all oocytes were routinely fertilized by
co-incubation per 100 with spermatozoa (frozen bull semen selected
by percoll gradient) at a final concentration of 10 6 sperm cells/ml for
20 h at 38.5ºC in fertilization medium, in a humidified 5% CO 2
incubator. Presumptive zygotes were cultured per 25 in 500µl droplets
of modified SOF medium with 5% FCS, under mineral oil for 8 days.
Results Binary logistic regression procedures were performed using
cleavage and blastocysts rate (blastocysts per oocytes matured) as
dependent variable, and treatment and replicate as independent
variables (SPSS 16.0). Cleavage and blastocyst rate were 70.5+7.5
and 29.4+3.1 in C, 80.6+8.5 and 35.5+11.3 in M, 40.8+12.9 and
4.9+3.4 in T, and 39.3+3.4 and 7.6+7.8 in TM groups, respectively.
Based on the odds ratio the cleavage rate decreases in the high
temperature group (by a factor of 0.27, P