Oocyte quality and heat shock proteins in oocytes from bovine breeds adapted to the tropics under different conditions of environmental thermal stress.

In order to evaluate the influence of thermal stress on physiological parameters, and the oocyte quality of Girolando (n = 12) and adapted Pantaneira (n = 12) cattle, twelve sessions of ultrasound guided follicular aspiration (OPU) were performed, between January and November 2014 (during dry (May-September) and rainy season (October-April) in Brazil). The recovered cumulus-oocyte complexes (COCs) were selected and classified, according to quality, immediately after OPU. The oocytes were then stored in 3% paraformaldehyd before conducting immunofluorescence analysis under confocal microscopy to identify HSP70 and 90 proteins. Before each OPU session, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. The black globe humidity index (BGHI) was calculated on the day of the OPUs and 90 days before each OPU session, and related to the thermal stress of the animals. The quality of oocytes from Girolando cattle, but not Pantaneira, showed a negative relationship with BGHI of 90 days before OPU. RT of both breeds did not exceed normal values for cattle below BGHI 95. BGHI variation on the day of OPU did not affect RF of the adapted Pantaneira breed (p = 0.3221). On the other hand, Girolando cattle showed a positive relationship between RF and BGHI (p = 0.0103). With increasing BGHI, the amount of HSP70 increased in Girolando oocytes, however, decreased in the Pantaneira breed. We have not observed a relationship between HSP 90 and BGHI, however Girolando cattle produced a greater amount of this protein in relation to the Pantaneira breed. In conclusion, higher BGHIs, 90 days before OPU session, negatively affect oocyte quality of Girolando cattle and positively affect oocyte quality of the Pantaneira breed. Higher BGHIs on the day of the OPU session negatively affected the respiratory frequency of the Girolando breed, and lead to a higher recruitment of HSP70 to protect oocyte maturation. The opposite pattern was observed for Pantaneira. In addition, Pantaneira cattle produced twice as much as HSP70 as Girolando cattle, suggesting that a natural higher production of this protein could be involved in the mechanisms of adaptation to heat conditions.

Efeito do estresse térmico calórico agudo e crônico sobre a qualidade oocitária de bovinos de raças adaptadas / Effect of acute and chronic caloric heat stress on oocyte quality of adapted breeds cattle

Nos trópicos, o uso de raças adaptadas tem sido uma estratégia para minimizar o efeito do estresse térmico calórico (ETC). No entanto, faltam informações que quantifiquem o estresse e o seu efeito sobre a reprodução dessas raças. O objetivo deste estudo foi avaliar a qualidade do oócito recuperado e alguns parâmetros fisiológicos indicadores de ETC em bovinos de raças adaptadas. Animais Bos taurus x Bos indicus (n=6) e Bos taurus (raça Pantaneira; n=12), localizados na região de transição entre o Cerrado e o Pantanal brasileiro, foram submetidos à aspiração folicular guiada por ultrassonografia (OPU) em diferentes condições climáticas. Foram realizadas oito sessões de OPU, com intervalo mínimo de sete dias e máximo de 54 dias entre as coletas. Para caracterização climática, foi realizado o cálculo do índice de temperatura e umidade (ITU). Foram quantificados os ITUs do dia da OPU, sete dias antes e 60 dias antes de cada sessão. Os parâmetros fisiológicos e a viabilidade oocitária de fêmeas das raças Girolando e Pantaneira não foram afetados negativamente por ITUs entre 72 e 78. O ETC crônico (60 dias) parece afetar a viabilidade oocitária de doadoras na raça Pantaneira quando ITU é superior a 75.(AU)

In tropical regions, the use of adapted breeds has been a strategy to minimize the effect of heat stress (HS) in cattle. However, information quantifying stress and its effect on reproduction of these breeds is lacking. The aim of this study was to evaluate the quality of the recovered oocyte and some physiological parameters that indicate HS in adapted breed. Bos taurus x Bos indicus (n=6) and Pantaneira (n=12) cows, located in the transition region between Cerrado and Brazilian Pantanal, underwent follicular aspiration guided by ultrasound (OPU) in different weather conditions. Eight sessions of OPU were carried out, with a minimum interval of 7 days and maximum 54 days between sessions. For weather characterization, the temperature and humidity index (THI) was calculated. THI of the day of OPU, 7 days before and 60 days before each session were calculated. The physiological parameters and oocyte viability of Girolando and Pantaneira cows were not negatively influenced under ITU between 72 and 78. The chronic HS (60 days)may affect the oocyte viability of Pantaneira donors when ITU is over 75.(AU)

Estimate of the population of preantral follicles in the ovaries of Bos taurus indicus and Bos taurus taurus cattle.

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.

Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors.

The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates.

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.