Synchronization of stage of follicle development before OPU improves embryo production in cows with large antral follicle counts.

In the present study, there was an evaluation of in vitro embryo production (IVEP) in Bos indicus donor cows with small or large antral follicle counts (AFCs) when there was synchronization of follicular dynamics among cows before ovum pick-up (OPU). Donor cows classified as having small or large AFC were submitted to OPU/IVEP program (Experiment-I) or had follicular-stage synchronization imposed before OPU/IVEP (Experiment-II). In Experiment-I, the cows with a large AFC had a greater (P < 0.01) mean of embryos developing to the blastocyst stage compared to those with a small AFC. In Experiment-II, percentage of viable oocytes/OPU were not affected (P = 0.33) by synchronization of follicular dynamics, but the AFC had an effect (P < 0.0001). There was an interaction (P = 0.01) indicating the larger AFC, with or without imposing of a synchronization treatment regimen, resulted in the most desirable outcome. The number of embryos was affected (P < 0.001) by follicular-stage synchronization and AFC, with there being an interaction (P = 0.002) with the most desirable results for the large AFC-synchronized group. Number of pregnancies was greater (P ≤ 0.02) for recipient females with embryos from synchronized donors and with a large AFC. There was an interaction (P = 0.03) with there being a greater pregnancy percentage for cows with synchronized follicular stages and the large AFC. Bos indicus donor with a large AFC when associated with the synchronization of stage of follicular dynamics pre-OPU results in improvement of the efficacy of IVEP.

Influence of cAMP modulator supplementation of in vitro culture medium on Bos taurus indicus embryos.

The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ±â€¯3.7) and Control (883 ±â€¯5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos.

Relationships between antral follicle count, body condition, and pregnancy rates after timed-AI in Bos indicus cattle.

An experiment was performed to evaluate the association between the antral follicle count (AFC) plus body condition score (BCS) and the pregnancy rate in Bos indicus undergoing timed artificial insemination (TAI). A total of 736 Nelore cows with BCSs ranging from 2 to 4 received a conventional protocol for TAI. On a random day of the estrous cycle (Day 0), all cows received an intravaginal P4 device and an intramuscular (i.m.) injection of 2.0 mg estradiol benzoate. On Day 8, the P4 device was removed, and 150 µg sodium D-cloprostenol, 300 IU equine chorionic gonadotrophin and 1.0 mg estradiol cypionate were administered by i.m. injection. TAI was performed 48 h after P4 device removal, and pregnancy diagnosis was performed by ultrasonography after 30 days. On Day 0, all cows were examined by ultrasonography to determine the AFC by counting the number of follicles >3 mm in diameter that were present in both ovaries and to evaluate the BCS (scale of 1-5). The cows were then classified based on their AFCs as those with low (≤10 follicles), intermediate (11-29 follicles) and high AFC (≥30 follicles). Furthermore, cows were classified as having low (≥2.0 to ≤ 2.9) and high (≥3.0 to ≤ 4.0) BCSs. The AFCs and BCSs were analyzed using the generalized linear model, and the pregnancy rate was assessed with the binary logistic regression model (P ≤ 0.05). The pregnancy rate was influenced (P < 0.05) by AFC and BCS classification and by interactions (P = 0.034) between these factors. Cows with a low AFC exhibited higher a pregnancy rate than did cows with a high or an intermediate AFC (57.7% a, 47.9% b and 49.7% b, P = 0.008). Low BCS resulted in a higher pregnancy rate than did high BCS (55.2% vs. 50.4%, P = 0.008). Cows with a high BCS and a low AFC had a higher pregnancy rate (P < 0.05) than did those with a high BCS and an intermediate or a high AFC (59.8%a, 48.0%b, and 38.0%b, respectively). An interaction (P < 0.05) was observed between the AFC and BCS, and the pregnancy rate decreased significantly in cows with an AFC > 30 and a BCS between 3 and 4. In conclusion, AFC and BCS classifications influence the pregnancy rate of Bos indicus beef cattle subjected to TAI. In addition, an important interaction between these factors was observed, namely, the lowest pregnancy rates were found in cows with high BCSs and high AFCs.

Injectable progesterone in timed artificial insemination programs in beef cows.

The aims of this study were I) to compare the follicular diameter, corpus luteum diameter and serum progesterone (P4) concentrations in cows treated with conventional protocol vs. injectable P4 protocol; II) to determine the serum P4 profile in ovariectomized heifers; and III) to compare pregnancy rate between protocols. In experiment I, multiparous cows received a protocol for ovulation synchronization with an intravaginal P4 device (n = 38; device + EB day 0; device removal + PGF2α + eCG + EC day 8) or injectable P4 (n = 38; injection + EB day 0; PGF2α + eCG + EC day 8). In experiment II, ovariectomized heifers (n = 8) were treated with injectable P4 and blood samples were collected to determine the serum P4 profile. In experiment III, multiparous cows were timed AI with two different P4 approaches, intravaginal P4 device (n = 48) or injectable P4 (n = 47). In the first experiment, cows treated with P4 device had higher (P < 0.05) diameter of dominant follicle after ovulation induction (11.6 ± 1.8 vs.10.3 ± 1.8 mm) and ovulation rate (97%, 37/38 vs. 47.3%, 18/38) than cows treated with injectable P4. But, the follicular growth daily was higher (P < 0.05) in cows treated with injectable P4 than intravaginal device (1.3 ± 0.4 vs. 1.0 ± 0.3 mm/day, respectively). In experiment II, the P4 concentration peak occurred within 48 hours (6.54 ng/mL) and decreased after 96 hours (P < 0.05) after P4 injection. In experiment III, cows with P4 device had higher (P < 0.05) pregnancy rate than the injectable P4 group (60.4 vs. 34.0%, respectively). These results demonstrate that although the intravaginal P4 devices showed a higher pregnancy rate, a protocol with injectable P4 represents an easier method and a promising alternative for TAI in cattle.

A new direct transfer protocol for cryopreserved IVF embryos.

The global demand for in vitro-produced (IVP) embryos of determined sex has greatly increased over the last decade. Efficient protocols for the direct transfer of IVP embryos are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified, or frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by ovum pickup (n = 112) from Girolando (Holstein-Gir) females (n = 36) were selected and submitted to IVM for 24 hours at 38.5 °C with 5% CO2 in air with saturated humidity. In vitro fertilization was performed with the thawed, sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and cultured for 7 days under the same IVM and IVF conditions of temperature and humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned for either the transferred fresh, vitrified/thawing, or frozen/directly embryo transfer into previously synchronized recipient females. Conception rates were analyzed by binomial logistic regression, and a probability level of P < 0.05 was considered significant. The conception rates were 51.35 ± 1.87% (133/259) for the fresh embryos, 35.89 ± 3.87% (84/234) for the vitrified embryos, and 40.19 ± 4.65% (125/311) for the frozen directly transferred embryos. These data demonstrate that IVP embryos with sexed semen could be directly transferred into recipient cows with similar conception rates to vitrified embryos. The comparison found that the use of frozen embryos in direct transfer provides easier logistics and a more practical approach for the transfer of IVP embryos on dairy farms.

Developmental block and programmed cell death in Bos indicus embryos: effects of protein supplementation source and developmental kinetics.

The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235) or Slow (n = 485) based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively). The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells) and those that reached the fourth cell cycle at 90 hpi (Slow). Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.