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Doctoral Thesis
DOI
https://doi.org/10.11606/T.10.2012.tde-14112013-104536
Document
Author
Full name
Roberta Machado Ferreira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2012
Supervisor
Committee
Baruselli, Pietro Sampaio (President)
Binelli, Mario
Lopes, Fabíola Freitas de Paula
Meirelles, Flavio Vieira
Wiltbank, Milo Charles
Title in Portuguese
A baixa fertilidade de vacas Holandesas (B. taurus) repetidoras de serviço durante o estresse térmico está relacionada à sua baixa competência oocitária
Keywords in Portuguese
DNA mitocondrial
Estresse térmico
Fertilidade
Qualidade oocitária
Repetidoras de serviço
Abstract in Portuguese
O objetivo desse estudo foi avaliar se a baixa fertilidade de vacas Holandesas repetidoras de serviço [RS; comparativamente a novilhas (NOV) e vacas próximas ao pico de lactação (PL)] está associada com comprometimento da qualidade oocitária e se esta condição é agravada pelo estresse térmico. Fêmeas das três categorias foram tratadas com o mesmo protocolo de sincronização da emergência de onda folicular. Cinco dias após o início do protocolo, a ovum pick-up (OPU) foi realizada e foram avaliados (Capítulo; Cap. 1) o número de folículos ovarianos, de oócitos totais e viáveis, temperatura retal (TR), temperatura de superfície cutânea (TC) e frequência respiratória (FR). Os oócitos viáveis foram utilizados para a produção in vitro de embriões (Cap. 2) e avaliações biomoleculares (Cap. 3). No Cap. 2, foram avaliados o desenvolvimento embrionário (taxa de clivagem, de blastocisto e de eclosão) e a qualidade dos embriões produzidos (número de células e frequência de núcleos fragmentados). No Cap. 3, realizou-se a extração de RNA e DNA de parte dos oócitos coletados para a quantificação relativa e absoluta de DNA mitocondrial (mtDNA) e a avaliação da expressão de genes relacionados à replicação/transcrição do mtDNA (PPARGC1A, NRF1, POLG, POLG2, TFAM e MT-CO1), à apoptose (BAX, BCL2 e ITM2B) e ao estresse térmico (HSP90AA1 e HSPA1AB). No Cap. 4, a taxa de concepção após IATF foi avaliada em ambas as estações do ano e nas três categorias quando o mesmo protocolo de sincronização para IATF e a mesma partida de sêmen foram utilizados. No Cap. 1, vacas RS e PL aumentaram sua FR e TR no verão em relação ao inverno (P<0,0001), enquanto as NOV mantiveram essas variáveis constantes em ambas as estações. Nas três categorias houve aumento (P<0,0001) da TC no verão, mas esta sempre foi superior (P<0,001) em vacas RS e PL do que nas NOV, independente da estação. O número de folículos e de oócitos totais e viáveis declinou nas RS e PL durante o verão, mantendo-se semelhante em NOV em ambas as estações. No Cap. 2, a taxa de clivagem foi semelhante entre categorias e estações. Já a taxa de blastocisto foi reduzida no verão nas três categorias, sendo essa queda mais acentuada nas RS. A taxa de eclosão e o número de células dos blastocistos foram menores no verão (independente de categoria). Menor número de células foi observado em embriões de RS e PL (independente da estação). Além disso, a porcentagem de núcleos fragmentados foi maior nos blastocistos das RS no verão. No Cap. 3, a expressão de ITM2B e BAX foi maior em RS durante o verão. Ainda, detectaram-se indícios da ativação de mecanismos pró-apoptóticos nos oócitos de RS (maior relação BAX/BCL-2) comparadas a PL e de mecanismos compensatórios da deficiência da função mitocondrial (menor conteúdo de mtDNA e maior expressão de PPARGCC1, NRF1, TFAM, POLG e POLG2) nas RS durante o verão em relação as demais categorias. No Cap. 4, menor taxa de concepção foi observada em RS e durante o verão. Os resultados geram evidências de que o baixo desempenho reprodutivo de vacas RS durante o verão deva estar relacionado ao comprometimento da qualidade de seus oócitos, demonstrado pelo seu reduzido conteúdo de mtDNA e elevada expressão de genes relacionados a replicação/transcrição do mtDNA, apoptose e síntese de chaperonas, culminado em baixa taxa de blastocisto e alta fragmentação nuclear destes.
Title in English
The low fertility of repeat-breeder Holstein (B. taurus) cows during summer heat stress is related to a low oocyte competence
Keywords in English
Fertility
Heat stress
Mitochondrial DNA
Oocyte quality
Repeat breeder
Abstract in English
The aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent forThe aims of the present study were to evaluate whether lower fertility of repeat-breeder (RB) Holstein cows [compared to peak lactation cows (PL) and heifers (H)] is associated with oocyte quality and whether this condition is aggravated by summer heat stress. Females of the three categories were treated with same protocol for synchronization of follicular wave emergence during summer and winter. Five days after the protocol onset, the ovum pick-up (OPU) was performed. The following variables were evaluated in Chapter 1: number of ovarian follicles before OPU, number of total and viable oocytes, rectal temperature (RT), cutaneous temperature (CT) and respiration rate (RR). Viable oocytes were sent for in vitro embryo production (Chapter 2) and bimolecular evaluation (Chapter 3). In Chapter 2, embryo development (rates of cleavage, blastocyst and hatching) and quality (total number of nuclei and frequency of nuclear fragmentation) were assessed. In Chapter 3, part of the oocytes were subjected to DNA and RNA extraction to allow relative and absolute quantification of mitochondrial DNA (mtDNA), and the evaluation of the expression of genes related to mtDNA replication/transcription (PPARGC1A, NRF1, POLG, POLG2, TFAM and MT-CO1), apoptosis (BAX, BCL2 and ITM2B) and heat stress (HSP90AA1 e HSPA1AB). In Chapter 4, RB, PL and H were subjected to same protocol for fixed-time AI using the same batch of semen of a single sire in order to evaluate their P/AI during summer and winter. In Chapter 1, RB and PL had increased (P<0.0001) RR and RT during summer compared to winter; while H maintained similar RR and RT in both seasons. CT was greater (P<0.0001) during summer than winter in all categories, but it was always higher (P<0.001) in RB and PL than H, regardless of season. The numbers of follicles and total and viable oocytes were lower in RB and PL during summer than winter, and maintained stable in H in both seasons. In Chapter 2, cleavage rate was similar among categories and between seasons. However, blastocyst rate was invariably reduced during summer, but more pronouced in RB. Hatching rate and the total number of nuclei were decreased during summer, regardless of category. Lower number of nuclei was observed in RB and PL embryos compared to H, regardless of the season. Furthermore, the percentage of fragmented nuclei was greater in RB blastocysts during the summer. In Chapter 3, expressions of ITM2B and BAX were greater in RB oocytes collected during summer. Also, the activation of pro-apoptotic mechanisms (greater BAX/BCL2 ratio) was suggested in RB heat stressed-oocytes compared with PL. Activation of compensatory mechanisms of deficient mitochondrial function (low number of copies of mtDNA and increased expression of PPARGCC1, NRF1, TFAM, POLG and POLG2) were also observed in RB heat stressed-oocytes compared with PL and H. In Charpter 4, lower P/AI was observed in RB and under summer heat stress. These data provide evidences that the lower reproduction performance observed in RB during heat stress may be due to impaired oocyte quality, as shown by their reduced mtDNA content and upregulation of several genes related to mtDNA replication/transcription, apoptosis and chaperones synthesis, resulting in lower blastocyst rate and higher nuclear fragmentation of embryos.
 
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Publishing Date
2014-02-17
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • FERREIRA, R.M., et al. The low fertility of repeat-breeder cows during summer heat stress is related to a low oocyte competence to develop into blastocysts [doi:10.3168/jds.2010-3904]. Journal of Dairy Science [online], 2011, vol. 94, p. 2383-2392.
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