Rodrigo de Andrade Ferrazza

Thermoregulatory responses of Holstein cows exposed to experimentally induced heat stress

Heat stress (HS) adversely influences productivity and welfare of dairy cattle. We hypothesized that the thermoregulatory mechanisms vary depending on the exposure time to HS, with a cumulative effect on the adaptive responses and thermal strain of the cow. To identify the effect of HS on adaptive thermoregulatory mechanisms and predictors of caloric balance, Holstein cows were housed in climate chambers and randomly distributed into thermoneutral (TN; n=12) or HS (n=12) treatments for 16 days. Vaginal temperature (VT), rectal temperature (Tre), respiratory rate (RR), heart rate (HR), and dry matter intake (DMI) were measured. The temperature and humidity under TN were 25.9±0.2°C and 73.0±0.8%, respectively, and under HS were 36.3±0.3°C and 60.9±0.9%, respectively. The RR of the HS cows increased immediately after exposure to heat and was higher (76.02±1.70bpm, p<0.001) than in the TN (39.70±0.71bpm). An increase in Tre (39.87±0.07°C in the HS vs. 38.56±0.03°C in the TN, p<0.001) and in VT (39.82±0.10°C in the HS vs. 38.26±0.03°C in the TN, p<0.001) followed the increase in RR. A decrease (p<0.05) in HR occurred in the HS (62.13±0.99bpm) compared with the TN (66.23±0.79bpm); however, the magnitude of the differences was not the same over time. The DMI was lower in HS cows from the third day (8.27±0.33kgd-1 in the HS vs. 14.03±0.29kgd-1 in the TN, p<0.001), and the reduction of DMI was strongly affected (r=-0.65) by changes in the temperature humidity index. The effect of environmental variables from the previous day on physiological parameters and DMI was more important than the immediate effect, and ambient temperature represented the most determinant factor for heat exchange. The difference in the responses to acute and chronic exposure to HS suggests an adaptive response. Thus, intense thermal stress strongly influence thermoregulatory mechanisms and the acclimation process depend critically on heat exposure time.

Effect of husbandry system on the technical and economic performance of dairy cattle

This study aimed to investigate the effects of husbandry system on the technical and economic performance of dairy farming. Samples included data from 61 dairy farms from the State of Minas Gerais, Brazil, which were collected between 2002 and 2011. Farms were categorized by type-pasturebased (PB), semi-confinement (SC), and confinement (C)-and technical and economic indexes were compared. In general, the results indicated indexes that are higher than the average for Brazilian farms but lower than those in other countries or technological farms in other Brazilian regions. Milk production was mainly determined by farm size rather than by productivity indexes. Components of the total and effective operational costs that were most significant were feeding followed by labor. The comparative analysis indicated that, although C systems have technical indexes that are superior to those of the PB and SC systems, economic performance was independent of the intensification level. Thus, pasture systems are potentially competitive, provided that the producers are efficient.

183 GENE EXPRESSION OF IN VITRO-MATURATED OOCYTES CAN BE MODULATED BY FOLLICLE EXOSOMES FROM COWS KEPT UNDER THERMONEUTRAL OR HEAT STRESS CONDITIONS

There are several intrafollicular agents that have the ability to interfere with the metabolism and development of the oocyte, among these we highlight the exosomes (EXO). Thus, the aim of this study was to evaluate the capacity of EXO extracted from the follicular fluid of cows kept under thermoneutral or heat stress conditions to modulate oocyte maturation in vitro. Twenty-four Holstein cows were subjected to the following treatments for 14 days: heat stress (HS; n=12), 38°C, 60% RH, temperature-humidity index=88; and thermo-neutral (TN; n=12), 24°C, 60% RH, temperature-humidity index=71. Cows had their follicles aspirated when their diameter reached 9 to 12mm; all follicles with this diameter were aspirated. All follicular fluid aspirated from cows subjected to HS or TN was pooled forming the groups (HS and TN). The EXO were obtained by ultracentrifugation of follicular fluid (120,000×g for 70min at 4°C, twice) and had their presence confirmed by transmission electron microscopy. Bos indicus cumulus-oocyte complexes (COC) collected from ovaries obtained in commercial slaughterhouse, were pooled in groups of 20 COC and randomly subjected to 1 of the following treatments: Control, matured in standard medium (TCM 199, supplemented with Earles salts, glutamine, NaHCO3, pyruvate, FSH, and amikacin); HS-EXO, matured in standard medium added with 10µL of a solution of follicular EXO from HS cows; and TN-EXO, matured in standard medium added with 10µL of a solution of follicular EXO from TN cows. The procedures were repeated 4 times, always with 20 COC per treatment in each replica. After 22h of maturation, COC were recovered and the expression of genes related to apoptosis protection (BCL2), cell viability (STAT3), cell maintenance (RPL15), oocyte competence (BMP15), oxidative stress (CPT1B), cumulus cell expansion (HAS2), cell cycle (CDCA8), and heat stress protection (HSF1) were assessed. Oocyte genes were differentially expressed according to the source of EXO. Groups were statistically analysed using ANOVA and Tukey tests. All genes, except CPT1B, showed lower expression in TN-EXO oocytes when compared with control and HS-EXO (P<0.05). CPT1B showed a higher expression in HS-EXO oocytes (P<0.05). The results showed that the addition of EXO from exogenous follicles can modulate the expression of oocytes genes related to cell viability and survival. The lower expression of these genes in TN-EXO suggested that the EXO obtained in TN conditions attenuate several genes related to the oocytes maturation and viability. Surprisingly, the control oocytes showed a similar gene expression pattern of the HS-EXO. In conclusion, EXO derived from follicular fluid of cows submitted to TN or HS conditions can modulate the gene expression of oocytes matured in vitro. These results open new perspectives for the use of theses EXO as a tool to increase the efficiency of in vitro oocyte maturation.

Quantitative proteomic profiling of bovine follicular fluid during follicle development

Abstract Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy nonlactating Holstein cows to determine the protein expression profile of FF at key stages of follicular development. Follicles were individually aspirated in vivo at predeviation (F1 ∼ 7.0 mm), deviation (F1 ∼ 8.5 mm), postdeviation (F1 ∼ 12.0 mm), and preovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FFs from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled with TMTsixplex and quantified using liquid chromatography-mass spectrometry/mass spectrometry. A total of 143 proteins were identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P < 0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Among the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function. Summary Sentence The protein dynamic changes in ovarian follicle microenvironment during the follicle development are critical for follicular maturation and influence follicular function in cows.