2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A005 STUDENT COMPETITION
OOCYTE RECOVER
OVERY IN QUEENS AFTER CONTR
ONTRACEPTIVE
TREATMENT
WITH DESLORELIN ACET
CETATE TE (SUPRELORIN
®)
Camila Louise Ackermann, , Eduardo Trevisol, Rodrigo Volpato, Ana Augusta Pagnano Derussi, Carlos Renato De Freitas Guatolini, Flávia Caroline Destro,
Nicole Ruas Sousa,
Tatiana Da Silv
ilva Rasc
ascado ado & Mar
aria Denise Lop
opes
UNESP, BOTUCATU, SP, BRAZIL.
GnRH agonists are used as reversible contraceptives in wildlife as an alternative to chemical contraceptives that cause adverse
effects and surgical contraception. However, the GnRH agonists reversibility is not completely understood, few studies describe spontaneous
estrus after use this contraceptive, and only one described induced ovulation (ACKERMANN et al., 2011; Anais XIX CBRA, 2011.). This
study aimed to evaluate the reversibility of deslorelin acetate in female cats through the induction of estrus, ovulation and subsequent oocyte
recovery. Ten pubescent female cats underwent vaginal cytology (CV) three times a week to identify the stages of the estrous cycle. When the
females presented CV characteristic of interestrus, evidenced by a percentage lower than 70% of superficial cells, the animals were sedated,
an implant of deslorelin acetate (4.7 mg Suprelorin ® ) was introduced in the subcutaneous tissue interscapular region. After insertion of the
implants, CV were performed every 48 h, and on day 90 the implants were removed. Ten days after implant removal, estrus and ovulation
were induced using 100 IU eCG and 84 h later, 100 IU hCG, respectively. Three days after hormone treatment, females were submitted to
ovariohysterectomy. The oviducts were washed with PBS at 38°C and the liquid recovered was observed under stereomicroscopic magnifying
glass for identification and enumeration of potential oocytes. The oocytes were isolated and stained with Hoechst 33342 and propidium iodide
for viability assessment in a fluorescent microscope Leica DM LB ® - blue filter (535 and 617 nm); oocytes were considered viable when
fluoresced blue. The corpora lutea (CL) were quantified, and to calculate the oocyte recovery rate the following formula was used: number of
oocytes recovered/number of CL x 100. The results were described as mean and standard deviation. After application of the implant, three
females showed CV characteristic of estrus during 4.3 ± 2.2 days. After this period, all 10 females showed CV characteristic of ovarian
quiescence. All females responded the induction of estrus and the ovulation protocol presenting CV and estrus behavior. On average, 13.1 ±
5.5 CL and 8.1 ± 5.5 oocytes were quantified per cat. The oocyte recovery rate was 56.8% ± 25.4 and all the stained oocytes were viable. We
conclude that there is reversibility of ovarian activity after implant of deslorelin acetate in domestic cats since estrus and ovulation were
induced successfully, allowing the recovery of viable oocytes.
Keywords: domestic cats, GnRH agonists, contraception.
A006 STUDENT COMPETITION
THE METHYLATION TION PATTERNS TERNS OF THE IGF2 AND IGF2R GENES IN BOVINE SPERMATOZ
OZOA ARE NOT AFFECTED BY FLOW
CYTOMETR
OMETRY SEX SORTING
José De Oliveira Carvalho Neto 1 , Valquíria Alice Michalczechen-Lacerda 2 , Roberto Sartori 3 , Fernanda Castro Rodrigues 4 , Otávio Bravim 5 , Maurício Machaim
Franco 6 & Margot Alves Nunes Dode 7
1,3,5,8
UNIVERSIDADE DE SÃO PAULO, PIRACICABA, SP, BRAZIL. 2,5,8 UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 4 UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA,
MG, BRAZIL. 6,7,8 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.
The process of sexing by flow cytometry exposes sperm to factors that can cause epigenetic changes, such as the methylation pattern
of the DNA. These changes may not affect development until the blastocyst stage but may explain later problems in embryo development and
failures in plancentation. These problems are common in cloned embryos, with higher embryonic loss between Days 30 and 90 of gestation likely
due to abnormal methylation of DNA. The objectives were to investigate the effect of sexing by flow cytometry on the methylation patterns of
the IGF2 and IGF2R genes. Each ejaculate of Nellore bulls (n = 4) was collected and separated into three fractions: non-sexed (NS), sexed for
X-sperm (SX), and sexed for Y-sperm (SY). Frozen-thawed sperm cells were used for genomic DNA isolation that was then treated with
sodium bisulfate in three different replicates. Treated DNA was used for PCR amplification, which was cloned, transformed into E. coli and
sequenced for analyses of the methylation patterns. For comparisons among groups, the Kruskal-Wallis test was performed (P = 0.05). No
differences in DNA methylation were found among NS (96.4 ± 0.4 and 7.2 ± 0.6%), SX (95.4 ± 0.4 and 8.0 ± 0.6%) and SY (96.9 ± 0.4 and
8.0 ± 0.6%) groups for IGF2 (n = 195 sequences) and IGF2R (n = 150 sequences) genes, respectively. However, for the IGF2R gene, a very
specific methylation pattern was observed in the 25th and 26th CpG sites, which were highly methylated in the NS (86.4 and 84.1%), SX (86.2
and 81.0%) and SY (89.6 and 62.5%) groups, differently from the pattern expected for this region in sperm. Furthermore, the percentage of
methylation on the 26th CpG site was lower in the SY group than NS and SX groups. In addition, after DNA sequencing, we identified 28 CpG
sites in each clone of the IGF2 gene, which was different from the 27 CpGs reported by other studies in Bos taurus. When the methylation pattern
between treatments was compared for each bull, bull 3 had a higher percentage of methylation in the SY group than in the SX, for the IGF2 gene.
However, when comparing bulls for each treatment, we observed an effect of bull in the methylation of the gene IGF2R for the SY group. Bulls
1 and 3 (6.1 ± 0.7 and 6.4 ± 0.6%) had a lower percentage of methylation in comparison to bulls 2 and 4 (9.9 ± 1.8 and 9.6 ± 1.3%). In conclusion,
the sex-sorting procedure by flow cytometry did not affect the DNA methylation patterns of the IGF2 and IGF2R genes. However, there was
a bull variation in the methylation pattern. Furthermore, a polymorphism was found in IGF2 gene, and a very specific methylation pattern was
observed in the IGF2R gene, probably due to an epigenetic characteristic in Bos indicus cattle. [Financial support. CNPq, FAPESP and
Embrapa].
Keywords: epigenetic, methylation pattern of the DNA, sexed sperm.
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