Hepatic mRNA expression of enzymes associated with progesterone metabolism and its impact on ovarian and endocrine responses in Nelore (Bos indicus) and Holstein (Bos taurus) heifers with differing feed intakes.

The aim of this study was to evaluate circulating progesterone concentration (P4), LH pulsatility and ovarian follicular dynamics in Nelore (B. indicus) and Holstein (B. taurus) heifers under high (HDMI) and low (LDMI) dry matter/energy intakes. In addition, the effects of dry matter/energy intake and breed on hepatic expression of six genes associated with P4 metabolism (AKR1C4, AKR1D1, CYP3A4, CYP2C19, SRD5A1, and SRD5A3) was evaluated. Heifers received an intravaginal P4 device (1 g), 2 mg of estradiol benzoate (EB) i.m. and 500 µg of PGF2α at the begging of the synchronization protocol (D0). Eight days later, the P4 device was removed and all heifers received 1 mg of EB 24h later. Regardless of dry matter/energy intake, the number of recruited follicles was greater in Nelore than in Holstein heifers. In contrast, the maximum diameter of the dominant follicle was greater in Holstein than in Nelore heifers. Circulating P4 concentrations were greater in Nelore than in Holstein from D2 to D9, and in heifers receiving LDMI than those receiving HDMI from D1 to D8 of hormonal protocol. In addition, Holstein heifers had greater LH pulsatility and area under the curve of LH peaks compared to Nelore heifers. However, no effects were observed for LH values between feed intake levels. Interestingly, Holstein heifers had higher expression of SRD5A1, AKR1C4, AKR1D1 than Nelore heifers; whereas, for Nelore heifers, only the expression of CYP3A4 was higher compared to Holstein heifers. In conclusion, there are important differences in the follicular dynamics, circulating P4 and LH pulsatility concentrations that need to be considered during synchronization protocols for Nelore and Holstein breeds. More importantly, these differences appear to be at least partially modulated by the level of feed intake and the contrasting enzyme system in the liver involved with P4 metabolism between these cattle breeds.

Associations of insulin resistance later in lactation on fertility of dairy cows.

The challenge of getting dairy cows pregnant during early lactation is a well-described, worldwide problem. However, specifically in farms with poor reproductive, nutritional, and environmental conditions/management, a low pregnancy rate during early lactation is followed inevitably by an increased number of nonpregnant cows after 150 days in milk, with even more difficulties to achieve pregnancy. Therefore, several studies were designed to understand and develop strategies to mitigate reduced fertility of cows during late lactation. Experiments were performed under tropical regions to determine metabolic status during lactation and association of stage of lactation on oocyte quality and fertility. Lactating cows with extended days not pregnant (e.g.,>150 days in milk) often had systemic metabolic alterations, including development of peripheral insulin resistance and various oocyte alterations, including reduced expression of genes encoding glucose transport proteins, reduced amounts of mtDNA, increased expression of mitochondria-related genes, and increased expression of apoptosis-related genes. Additionally, in vitro embryo production and pregnancy per AI were lower in late- versus early-lactation cows in some but not all studies. Notwithstanding, when a normal embryo was transferred to a cow in late lactation, the pregnancy per transfer was reasonable, reinforcing the assertion that fertility problems in late-lactation cows may be associated with oocyte quality, fertilization, and/or failure of early embryo development. In conclusion, insulin resistance may reduce oocyte competence and consequently fertility in late-lactation dairy cows.

Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

Modulation of periovulatory endocrine profiles in beef cows: consequences for endometrial glucose transporters and uterine fluid glucose levels.

In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus.

Organic selenium supplementation increases PHGPx but does not improve viability in chilled boar semen.

This study evaluated the effects of dietary organic selenium (Se) on viability of chilled boar semen. Twelve boars were divided into three groups: control (CON), 0.3 mg kg(-1) sodium selenite; inorganic (INO), 0.5 mg kg(-1) sodium selenite and organic (ORG), 0.5 mg kg(-1) Se yeast. The experiment was conducted within 10 weeks, and analysis was performed fortnightly, in storage semen by 72 h. No effect was observed on motility; however, straightness and linearity percentages were higher (P < 0.05) in the animals receiving CON diet compared with INO group. Percentages of cells with both plasma and acrosomal intact membranes, lipidic membrane peroxidation and mitochondrial membrane potential were similar on all treatments. Animals receiving CON diet presented higher (P < 0.05) values of ATP when compared with INO group. The PHGPx was higher (P < 0.05) in animals that received ORG in comparison with INO group. In conclusion, organic selenium supplementation increases PHGPx but does not improve chilled semen viability in 72 h.

The influence of morphology, follicle size and Bcl-2 and Bax transcripts on the developmental competence of bovine oocytes.

This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1-3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6-9 mm (95.6; 41.1%), but similar (p > 0.05) to 3-6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1-3 mm (Bax; 1.0 and Bcl-2; 1.0), 3-6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6-9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.

The low fertility of repeat-breeder cows during summer heat stress is related to a low oocyte competence to develop into blastocysts.

It was hypothesized the lower fertility of repeat-breeder (RB) Holstein cows is associated with oocyte quality and this negative effect is enhanced during summer heat stress (HS). During the summer and the winter, heifers (H; n=36 and 34, respectively), peak-lactation (PL; n=37 and 32, respectively), and RB (n=36 and 31, respectively) Holstein cows were subjected to ovum retrieval to assess oocyte recovery, in vitro embryonic developmental rates, and blastocyst quality [terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and total cell number]. The environmental temperature and humidity, respiration rate, and cutaneous and rectal temperatures were recorded in both seasons. The summer HS increased the respiration rate and the rectal temperature of PL and RB cows, and increased the cutaneous temperature and lowered the in vitro embryo production of Holstein cows and heifers. Although cleavage rate was similar among groups [H=51.7% ± 4.5 (n=375), PL=37.9% ± 5.1 (n=390), RB=41.9% ± 4.5 (n=666)], blastocyst rate was compromised by HS, especially in RB cows [H=30.3% ± 4.8 (n=244) vs. 23.3% ± 6.4 (n=150), PL=22.0% ± 4.7 (n=191) vs. 14.6% ± 7.6 (n=103), RB=22.5% ± 5.4 (n=413) vs. 7.9% ± 4.3 (n=177)]. Moreover, the fragmentation rate of RB blastocysts was enhanced during the summer, compared with winter [4.9% ± 0.7 (n=14) vs. 2.2% ± 0.2 (n=78)] and other groups [H=2.5% ± 0.7 (n=13), and PL=2.7% ± 0.6 (n=14)] suggesting that the association of RB fertility problems and summer HS may potentially impair oocyte quality. Our findings provide evidence of a greater sensitivity of RB oocytes to summer HS.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin.

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 microg/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta Psi(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta Psi(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Characterization of mitochondrial genotypes in the foundation herd of the Canchim beef cattle breed.

The Canchim (5/8 Charolais + 3/8 Zebu) beef cattle breed was developed at Southeast-Embrapa Cattle to take advantage of hybrid vigor and to combine the higher growth rate and beef quality of Charolais with tropical adaptations of Zebu. The development of three lineages (old, new, and crossbred) has increased its genetic basis. The genotypic origin (Bos taurus or Bos indicus) of the mitochondrial DNA (mtDNA) of the Canchim breed was unknown. We characterized the mtDNA genotype of this founder herd by allele-specific polymerase chain reaction. The 173 founder Zebu females (62 Indubrasil, 3 Guzerat, and 108 Nellore) and their 6749 offspring were identified. The frequency of B. indicus mtDNA ranged from 1.15 to 2.05% among the descendants (n= 6404) of each maternal line with available DNA, and among animals that were alive (n= 689) in December 2007 among the three lineages. Though mtDNA characterization can be used to direct animal selection, the low frequency of B. indicus mtDNA impairs the evaluation of its effects on production traits in these animals. The high prevalence of B. taurus mtDNA in Canchim proves that the founder Zebu females from the Indubrasil, Guzerat and Nellore breeds were obtained from crosses of Zebu sires with local B. taurus dams.

Increase in mitochondrial DNA quantity and impairment of oxidative phosphorylation in bovine fibroblast cells treated with ethidium bromide for 15 passages in culture

Bovine fetal fibroblast cells were treated with ethidium bromide at a low concentration for 15 passages in culture to determine its effect on mitochondrial DNA copy number and on cell metabolism. Mitochondrial membrane potential and lactate production were estimated in order to characterize cell metabolism. In addition, mitochondrial DNA ND5 in proportion to a nuclear gene (luteinizing hormone receptor) was determined at the 1st, 2nd, 3rd, 10th, and 15th passages using semi-quantitative PCR amplification. Treated cells showed a lower mitochondrial membrane potential and higher levels of lactate production compared with control cells. However, the mitochondrial DNA/nuclear DNA ratio was higher in treated cells compared with control cells at the 10th and 15th passages. This ratio changed between the 3rd and 10th passages. Despite a clear impairment in mitochondrial function, ethidium bromide treatment did not lead to mitochondrial DNA depletion. It is possible that in response to a lower synthesis of ATP, due to an impairment in oxidative phosphorylation, treated cells develop a mechanism to resist the ethidium bromide effect on mtDNA replication, resulting in an increase in mitochondrial DNA copy number.