Sperm subpopulations evaluations on dogs ejaculate aiming fertility and cryopreservation analysis

• Grant number: 13/02050-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2013
• End date: July 31, 2016
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Maria Denise Lopes
• Grantee: Carmen Cecilia Sicherle
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

The aim of this study is to identify animals with high and low fertility and observe its resistance against cryopreservation through cluster analysis of sperm subpopulations. First the semen will be evaluated to identify and study sperm subpopulations (SE), trough the computer-assisted sperm analysis (CASA); check the cell membrane integrity; quantification of sperm apoptosis; capacitation status and acrosomal reaction during the cryopreservation process. Two experimental steps are proposed. In Experiment 1 sperm subpopulation will be determined during the cryopreservation process of the semen of 5 dogs, using 4 ejaculates from each animal, on fresh, cooled and thawed semen. Sperm kinematics will be checked by CASA and membrane integrity by flow cytometer by the combination of the two fluorescent probes propidium iodide (IP) and conjugate pisum sativum aglutinine and fluoresceine isocianete (FITC-PSA). The apoptosis test by Anexine-V and capacitation status by combination of the fluorcromes merocianine-540 (M540) and Yo-Pro 1. With data analysis we expect to separate dogs in two groups with high and low fertility (HIF and LOF). Among 5 dogs will be choose 1 of HIF and 1 of LOF those to be used on the 2th experiment. Experiment 2: with data generated from the first of the sperm subpopulations infer about the potential of a sperm gradient separation in recuperate sperm subpopulation of high fertilising potential and evaluate its application on artificial insemination. For this purpose the PureSperm® gradient with density of 80 and 40 will be done following the methodology of Dourado et al., 2011. Fourty (40) bitchies will be inseminated for fertilizing results comparing among dogs of high fertility (HIFS/HIFNS n = 20) and low fertility (LOFS/LOFNS n = 20) using (HIFS and LOFS) or not (HIFNS and LOFNS) the sperm separation method. The transcervical artificial inseminations will be performed with the aid of a rigid endoscope to reach and pass trough the cervical os. After 10/12.days from ovulation the ovarosalpingoestretomie (OSH) will be performed as the recovery and counting of embryos. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CARMEN CECILIA SICHERLE; FABIANA FERREIRA DE SOUZA; CAMILA DE PAULA FREITAS-DELLAQUA; GABRIELE BARROS MOTHÉ; CARLOS ROBERTO PADOVANI; FREDERICO OZANAM PAPA; MARIA DENISE LOPES. Effects of the cryopreservation process on dog sperm integrity. Animal Reproduction, v. 17, n. 1, 2020-04-03. (13/02050-5)

SICHERLE, CARMEN CECILIA; DE SOUZA, FABIANA FERREIRA; FREITAS-DELL'AQUA, CAMILA DE PAULA; MOTHE, GABRIELE BARROS; PADOVANI, CARLOS ROBERTO; PAPA, FREDERICO OZANAM; LOPES, MARIA DENISE. Effects of the cryopreservation process on dog sperm integrity. ANIMAL REPRODUCTION, v. 17, n. 1, p. 10-pg., 2020-01-01. (13/02050-5)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

SICHERLE, Carmen Cecilia. Frozen semen and artificial insemination in dogs. 2016. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária e Zootecnia. Botucatu Botucatu.