Vascular and morphological features of the corpus luteum 12 to 20 days after timed artificial insemination in dairy cattle.

Our objective was to retrospectively compare pregnant versus nonpregnant cattle in terms of vascular and morphometric changes in corpora lutea between d 12 and 20 following timed artificial insemination (TAI). Crossbred (Gir × Holstein) lactating dairy cows (n = 136) and heifers (n = 111) were bred after synchronizing ovulations using an estradiol plus progesterone (P4)-based protocol. Corpus luteum (CL) characteristics (area, echotexture, blood flow) were recorded at 48-h intervals from d 12 to 20 following TAI using an ultrasound equipped with color Doppler. Blood samples were collected to determine CL function (plasma P4). Pregnancy diagnosis was performed at d 30. Quantitative assessment of colored pixels within the CL was performed using ImageJ software (National Institutes of Health, Bethesda, MD) and echotexture was quantified using custom software. Continuous variables such as luteal tissue area (LTA), CL blood flow (CLBF), adjusted CLBF (ratio LTA:CLBF), mean pixel value (MPV), pixel heterogeneity (HETER), and plasma P4 were analyzed retrospectively as repeated measures (d 12 to 20) in pregnant versus nonpregnant females using PROC MIXED (SAS Institute Inc., Cary, NC). Main effects were pregnancy status, day of cycle, and their interaction. Further analyses used only data from d 16, because this was the earliest time point of deviation between CLBF of pregnant and nonpregnant animals. We created quartiles for each variable and calculated the risk of pregnancy within quartile. Differences were determined using the chi-squared test. Plasma P4 was significantly higher in prospective pregnant versus nonpregnant cattle on d 18 and 20, whereas LTA differed only on d 20. On d 16, CLBF and adjusted CLBF diverged between pregnant and nonpregnant, followed by a progressive reduction in the latter until d 20. Mean pixel value was not affected by pregnancy status, but HETER was lower on d 20 in pregnant than in nonpregnant cattle. Likelihood of pregnancy increased from quartile (Q)1 (lowest values) to Q4 (highest) of CLBF (Q4 vs. Q1, odds ratio = 32.8, 95% confidence interval: 9.6 to 112.1) and adjusted CLBF [Q4 vs. Q1, odds ratio = 25.4, 95% confidence interval: 8.1 to 80.4), whereas a lower risk of pregnancy was observed only for animals within Q1 of plasma P4 [Q4 vs. Q1, odds ratio = 3.1, 95% confidence interval: 1.3 to 7.2). Day 16 quartiles of LTA, MPV, and HETER did not affect odds of pregnancy. In conclusion, we identified distinct CLBF patterns as early as 16 d after TAI and confirmed that CL function is lost by a reduction in blood flow, which precedes physical regression.

Biocompatibility assessment of fibrous nanomaterials in mammalian embryos.

Currently there is a growing interest in the use of nanotechnology in reproductive medicine and reproductive biology. However, their toxic effects on mammalian embryos remain poorly understood. In this work, we evaluate the biocompatibility of two fibrous nanomaterials (NMs): cotton cellulose nanofibers (CNF) and carboxylated multiwalled carbon nanotubes (MWCNT-COOH), by performing an investigation of the embryonic development, gene expression (biomarkers focused on cell stress, apoptosis and totipotency) and in situ apoptosis in bovine embryos. Exposure to NMs did not interfere in preimplantation development or in the incidence of apoptosis in the bovine embryo, but they did affect the gene expression. The results presented are important for an understanding of the toxicity of cotton CNF and MWCNT-COOH on mammalian embryos. To our knowledge, we report the first evaluation of biocompatibility between these NMs on preimplantation embryos, which may open a new window for reproductive biomedical applications.

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle.

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus x Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n=12) or during a shortened cycle, with luteolysis induction 10d after estrus (Experiment 2; n=6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10d after estrus (P<0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r=0.86; P<0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24h compared with 72 h; P<0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P>0.70), but a day effect on this attribute was observed in Experiment 2 (P=0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r=-0.63; P<0.05), but not in Experiment 2 (r=-0.28; P>0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P<0.009) and luteolysis-induced (Experiment 2; P<0.05) estrous cycles (P<0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r=-0.71 and -0.58 in Experiments 1 and 2, respectively; P<0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.