The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

Valuable female cattle are continuously subject to follicular puncture (ovum pickup -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 × 10 6 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 OPUlesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

RESUMO O Brasil atualmente é detentor do terceiro maior rebanho de vacas leiteiras do mundo, composto, em sua maioria, de animais mestiços F1 (Gir X Holandês), os quais são bem adaptados às áreas tropicais. O objetivo do presente estudo foi investigar a eficiência da ovum pick-up (OPU) e da produção in vitro de embriões oriundos de doadoras Girolando com sêmen sexado de touros provenientes de duas raças, Gir e Holandesa. Foram utilizados dados referentes a 232 animais de diversos rebanhos, totalizando 4334 oócitos recuperados. Os oócitos foram classificados, e as estruturas viáveis (GI, GII e GIII) foram utilizadas para produção in vitro de embriões (PIVE). Foi observada uma média de oócitos recuperados para as doadoras F1 de 18,14±1,33. Além disso, notou-se que não houve diferença entre os dois cruzamentos utilizados, considerando média de total de embriões (4,96±0,40 e 6,56±0,76) para o cruzamento F1 X HPB (Holandês preto e branco) e F1 X Gir, respectivamente. Portanto, as doadoras F1 apresentaram potencial como doadoras de oócitos no sistema de produção in vitro de embriões, independentemente da raça do touro cujo sêmen foi utilizado.

No abstract

Recent advances in image technology, including significant gains in spatial resolution, have made realtime sequential ovarian evaluations possible in small rodents, allowing longitudinal (continued) studies of the ovarian cycle and reducing the required number of experimental animals. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using high-resolution ultrasound technology. Female mice (n ¼ 15) received a 5 IU intraperitoneal injection of equine chorionic gonadotropin (eCG) and 48 h later a 5 IU injection of human chorionic gonadotropin (hCG), and were allowed to mate thereafter. In experiment 1, animals (n ¼ 7) were evaluated every 6 h, from 3 to 51 h after eCG injection, with an ultrasound biomicroscopy (UBM) equipped with a realtime 45 MHz microvisualization probe (RMV 707b). The ovaries were identified and follicular population quantified, and follicles were classified according to the diameter as small ( 449 mm) or large (!450 mm). A significant change in the distribution of follicle population according to category was observed only 45 h after eCG injection (P , 0.05). In experiment 2, animals (n ¼ 8) were evaluated every 2 h, from 2 h to 10 h after hCG treatment. The largest follicles reached a maximum size (596.7 + 106.0 mm) 5.8 + 2.3 h after hCG injection. As expected, the population of large follicles decreased thereafter, indicating the progress of ovulations, but large follicles were still detected late after treatment (10.1 + 1.1 h). In conclusion, UBM can be used to evaluate follicle dynamics in superstimulated mice (C57BL/6 and BALB/c); significant changes in follicle distribution only occur at later stages after eCG stimulation; and hCG-induced ovulations may not occur synchronously in mice.

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.

The objective of the present study was to evaluate the effect of two diets with different energy levels and two genetic groups (3/4 and 7/8 Holstein × Gir (HG) cows) on the metabolic and hormonal changes and on the production profile of oocytes and embryos in dairy cows during the early postpartum period. The concentrations of oestrogen, progesterone, glucose, insulin, insulin-like growth factor, urea and non-esterified fatty acids in follicular fluid, as well as blood plasma concentrations of glucose, insulin, urea and non-esterified fatty acids, were evaluated. Oocyte collection was performed every 14 days after parturition. After classification, the oocytes were submitted to in vitro embryo production and cleavage, and blastocyst rates were evaluated. Five days after oocyte collection, the dominant follicle was measured and punctured for follicular fluid retrieval. The high-energy diet increased plasmatic insulin and glucose. The 3/4 HG cows presented a higher plasmatic concentration of insulin, glucose and urea. The hormonal and metabolic changes in plasma were not observed in the follicular fluid. The follicular fluid concentration of IGF-I was increased in cows fed the high-energy diet as well as in the 3/4 HG cows. A higher number of total and viable oocytes was recovered in the 3/4 HG cows, but the 7/8 HG cows had a higher cleavage rate. In conclusion, the high-energy diet was more efficient in maintaining the energy status of crossbred cows, as evidenced by their plasma metabolites and follicular fluid, and 3/4 HG cows were more efficient than 7/8 HG cows at producing oocytes in the early postpartum period.

The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.