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.

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 in the HS vs. 14.03±0.29kgd 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.

Introduction. During decades, milk production in the Nariño state has depended on the Holstein breed. For this reason, it is necessary to evaluate a model of milk production that allows to decrease production costs and in turn improves the compositional quality of the milk. Objective. This study aimed to compare milk production and compositional quality of Holstein (HO) and the crossbreed between Kiwi Cross (KC) x HO. Materials and methods: Monthly milk production in HO cows (n=30) and the ones from the F1 of the KC x HO cross (n=40), was measured by adjusting the day in milk (DIM) and milk production by third of lactation, fat, protein and total solids. For the period between October 2016 and May 2017, 9,809 dairy production records were analyzed. Results: Maximum production was 25.8 ± 0.53 vs. 23.2 ± 0.53 l day-1 for HO vs. KC cows (p<0.05), respectively. The DIM was not different; however, in days 60, 90, 150, 180, 210 and 240 the HO group produced more milk than the KC group, with a persistence in the lactation peak until day 60, and from that point onwards milk production showed decreasing rates in both groups. Furthermore, the production per third of lactation was higher (p<0.05) in the first third compared to the second and third periods for HO (13.6±0.56 vs. 11.3±0.5723 and 9.9±0.47 l day-1, respectively) and KC (12.8±0.4505 vs. 10.6±0.66 and 9.5 ± 1.69 l day-1, respectively). Fat content was higher (p<0.05) in KC compared to HO in week one, three and five (4±0.07, 4±0.07, 4±0.07 vs. 3.6±0.12, 3.6±0.11, 3.7±0.09 %, respectively); likewise, protein in week one and four was higher in the KC group compared to HO (3.3±0.04 vs. 3.1±0.05 %; p<0.05). Total solids were 13.3±0.17 vs. 12.5±0.23% (p<0.05) for KC and HO cows, respectively in weeks two and five. Conclusion: Milk production in KC and HO was similar; however, KC improved performance in compositional milk quality, increasing the percentages of fat, protein and consequently, total solids.

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Background Feline obstructive disease of the lower urinary tract (FLUTD) is a common pathologic condition of cats. It can be related to sterile inflammation, which leads to acute impairment of renal function and the accumulation of electrolytes and acid‐base imbalance. Acute‐phase proteins (APPs) are biomarkers of tissue damage from inflammation that assist in monitoring treatment and prognosis. Objective Monitoring the inflammatory processes of obstructive feline lower urinary tract disease through the determination of plasma fibrinogen concentrations and serum concentrations of the acute‐phase proteins, serum amyloid A (SAA), alpha‐1‐acid glycoprotein (AGP), and albumin. Methodology Twenty‐five male cats were included in this study. They were divided into two experimental groups: a control group (CG) and an obstruction group (OG). There were 8 healthy cats in the CG group and 17 cats with obstructive FLUTD in the OG group. APP measurements were conducted using ELISA kits. Samples were collected for APP analyses, serum biochemical assays, urinalyses, and urine protein: creatinine ratio calculations at diagnosis, before urethral clearance (H0), and 12 (H12), 24 (H24), and 48 (H48) hours after urethral clearance from cats in the OG group. Samples were collected once from cats in the CG group cats. Results At H0, we found positive correlations of SAA, AGP, and fibrinogen with urea and creatinine, and negative correlations of albumin with hematuria, SAA, and potassium. At H48, we found positive correlations between SAA and AGP, AGP and urea, fibrinogen and urea, fibrinogen and creatinine, fibrinogen and AGP, and fibrinogen and SAA. In addition, a negative correlation of albumin with urea and creatinine was observed. Conclusions Serum amyloid A, AGP, fibrinogen, and albumin could be used as biomarkers of inflammatory processes in cats with obstructive FLUTD.

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 12 mm; 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 70 min 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 Earle’s salts, glutamine, NaHCO3, pyruvate, FSH, and amikacin); HS-EXO, matured in standard medium added with 10 µL of a solution of follicular EXO from HS cows; and TN-EXO, matured in standard medium added with 10 µL 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 22 h 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. Financial support from FAPESP #12/18297–7.

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