Actions of CSF2 and DKK1 on bovine embryo development and pregnancy outcomes are affected by composition of embryo culture medium.

Procedures for in vitro embryo production in cattle have not been optimized. In the current experiment, we utilized a 3 × 3 factorial design to test whether the proportion of embryos becoming blastocysts in culture and the pregnancy rate after embryo transfer are affected by type of serum in the medium [no serum; 3% (v/v) KnockOut Serum Replacement (SR); 3% (v/v) fetal bovine serum (FBS)] and addition of specific embryokines [vehicle; 10 ng/mL colony stimulating factor 2 (CSF2); 100 ng/mL dickkopf related protein 1 (DKK1)] at day 5 of culture. Embryos were produced using abattoir-derived ovaries and Y-sorted semen from two Angus sires. The percent of putative zygotes and cleaved embryos becoming blastocysts was improved by SR and FBS. Pregnancy rate at day 30 was determined for 1426 Nelore recipients and calving rate for 266 recipients. In the absence of CSF2 or DKK1, pregnancy rates were lower for embryos cultured with SR or FBS. CSF2 and DKK1 reduced pregnancy rate for embryos cultured without serum but had no detrimental effect in the SR or FBS groups. Indeed, CSF2 blocked the negative effect of FBS on pregnancy rate. Data on birth weights were available for 67 bull calves. There were no effects of treatment. The sire used to produce embryos had significant and large effects on development to the blastocyst stage, pregnancy rate at day 30, calving rate and pregnancy loss between day 30 and calving. Results indicate that (1) SR and FBS can improve embryonic development in vitro while also compromising competence of embryos to survive after transfer, (2) actions of CSF2 and DKK1 depend upon other characteristics of the embryo production system, and (3) sire can have a large effect on embryonic development before and after transfer.

Intraovarian injection of mesenchymal stem cells improves oocyte yield and in vitro embryo production in a bovine model of fertility loss.

Valuable female cattle are continuously subject to follicular puncture (ovum pick-up - OPU). This technique is commonly used for in-vitro embryo production, but may result in ovarian lesion. Mesenchymal stem cells (MSC) ameliorate the function of injured tissues, but their use to treat ovarian lesions in cattle has not been established. We investigated whether a local injection of MSC would reduce the negative effects of repeated OPU under acute and chronic scenarios in bovines. First, we performed four OPU sessions and injected 2.5 × 106 MSCs immediately after the 4th OPU procedure (n = 5). The treated organs (right ovary) were compared to their saline-treated counterparts (left), and presented superior production of oocytes and embryos in the three following OPU sessions (P < 0.05). Then, cows with progressive fertility loss went through three OPU sessions. Animals received MSC, saline, or MSC + FSH in both ovaries after the first OPU. In the two following OPU sessions, the MSC and MSC + FSH - treated groups failed to present any significant alteration in the number of oocytes and embryos compared to saline-treated animals. Thus, MSC have beneficial effects on the fertility of OPU-lesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

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.

Caracterização de seqüelas subseqüentes à punção folicular em bovinos / Characterization of tissue damages after ovum pick-up in bovine

Objetivaram-se avaliar lesões no trato genital em animais utilizados como doadores de oócitos. Foram examinadas vacas da raça Gir (n=20) que participaram de diferentes experimentos envolvendo a técnica de punção folicular. Ao término de cada período experimental, os animais foram examinados por palpação retal, ultra-sonografia e vaginoscopia. Um grupo de vacas (n=8) foi selecionado para a caracterização histopatológica das lesões ovarianas. Os ovários foram retirados cirurgicamente, fixados e processados, e as lâminas obtidas coradas e analisadas. Foram avaliados animais submetidos a entre nove e 42 sessões de punção, e que produziram entre 10 e 719 oócitos. Observou-se uma baixa prevalência (5,26 por cento) de processos inflamatórios na mucosa do fundo de saco vaginal. A ocorrência de aderências apresentou uma correlação positiva com o número de sessões de punção a que o animal foi submetido (r=0,31; P<0,05), mas não com o total de oócitos recuperados (r=0,03; P>0,05). Tendência oposta foi observada em relação à fibrose ovariana, que apresentou uma alta correlação com o número de oócitos recuperados (r=0,53; P<0,05). A avaliação histopatológica demonstrou a presença de tecido cicatricial, infiltrados de células inflamatórias e presença de tecido luteal disperso no estroma ovariano.