Characterization of Anti-Müllerian hormone concentrations in domestic dogs and gray wolves (Canis lupus baileyi)

• Grant number: 17/06047-0
• Support Opportunities: Regular Research Grants
• Start date: August 01, 2017
• End date: July 31, 2019
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Maria Denise Lopes
• Grantee: Maria Denise Lopes
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated researchers: Andressa Dalmazzo

Abstract

We aim to determine antimullerian hormone levels in Gray Wolves (Canis lupus baileyi) (experiment 1) and in domestic dogs (experiment 2), and to use AMH blood levels, in domestic dogs, as a tool for choose ovary donors, for cryopreservation, as an experimental model for wolves and to evaluate AMH production in fresh and vitrified/warmed ovarian cortex cultured in vitro (experiment 3). Experiment 1: Characterization of AMH blood levels in gray wolves. Blood from seven gray wolves will be collected during reproductive season (Winter) and non-reproductive season (Summer), during 2 consecutive years. During this period a questionnaire will be filled to register the reproductive history aiming to correlate these information with AMH concentration. Experiment 2: Characterization of AMH blood levels in domestic dogs. Blood samples from 90 dogs will be collected. The dogs will be divided in 6 experimental groups: pre-pubertal (G1, n=15), young bitches (1st and 2nd estrous) (G2, n=15), adults (G3, n=15), pregnant (G4, n=15), old (more than 9 years) (G5, n=15) and spayed (G6, n=15). For G3 and G4, blood will be collected once during each one of the estrous phase (proestrous, estrous, diestrus and anestrous) aiming to evaluate possible variations among the cycle. All samples will be centrifuged and serum will be stored at -20 oC until analysis. AMH will be evaluated using ELISA kit (AnshLab, Texas, EUA). Experimento 3: Produção de AMH em cultura in vitro de ovários frescos e após descongelação de cadelas. Twenty bitches will be spayed and will have a blood sample collected. Ovaries will be isolated and will have their cortex sliced in four pieces. One fresh piece will be used for in vitro culture and one will be use to histological evaluation. The other two pieces will be submitted to cryopreservation. The slices will be warmed, for that the pieces will be immersed in a Petri dish containing thawing solution thereafter it will be transferred to a petri dish containing Sucrose solution. Both fresh and thawed samples will be cut into smaller pieces and placed in 96 well plate, witch well containing 200 µL of growth media. Plates will be maintained at 5% CO2 and 38.5ºC. Every other day 100 µL of media will be removed and stored at -20oC for AMH, P4 and estrogen assay, the same volume will be replaced with fresh media. After 7 days of culture, sample will be removed and processed to histological analysis. Hormonal assay will be performed to determine AMH in in vitro culture media and serum by ELISA. (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)

ACKERMANN, C. L.; KOZLOWSKI, C. P.; CALLAHAN, M.; BAUMAN, K.; CLAWITTER, H.; ASA, C. S.; LOPES, M. D.. Anti-Mullerian hormone concentration in female gray wolf (Canis lupus) - preliminary results. REPRODUCTION IN DOMESTIC ANIMALS, v. 54, p. 2-pg., 2019-06-01. (17/06047-0)