E. Nogueira

Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants

The effects of intracellular (cysteine and β-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification-thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%-56.4%) or the timing of supplementation (41.7%-55.4%) compared with control (48.7%; P>0.05). Similarly, antioxidants and the moment of supplementation did not affect (P>0.05) the total number of blastomeres (86.2-90.5 and 84.4-90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P<0.05) in groups supplemented during IVM (1.7%), IVC (2.0%) or both (1.8%) compared with control (4.3%). Intracellular ROS levels measured in Day 7 blastocysts were reduced (P<0.05) in all groups (0.60-0.78), with the exception of the group supplemented with β-mercaptoethanol during IVC (0.88), which did not differ (P>0.05) from that in the control group (1.00). Re-expansion rates were not affected (P>0.05) by the treatments (50.0%-93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.

Sugar cane treated with calcium hydroxide in diet for cattle: intake, digestibility of nutrients and ingestive behaviour

The objective of this work was to evaluate, in bovines, the intake and apparent digestibility of nutrients, ingestive behaviour and content of total digestible nutrients (TDN) of diets based on sugar cane treated with calcium hydroxide. It was used four cows with average body weight of 412.06 kg ± 37.3 kg. The study included inclusion of calcium hydroxide at the doses 0, 8, 16 and 24 g/kg of sugar cane in natural matter basis fed in diets with 65% of roughage and 35% on concentrate, based on dry matter (DM). It was used a 4 × 4 Latin square experimental design with four treatments and four periods. Calcium hydroxide doses affected nutrient intake: DM intake was 7.88 kg/day at the dose of 14.99 g of calcium hydroxide, organic matter (OM) intake was 6.75 kg/day at 10.94 g; intake of neutral detergent fiber (NDF) of 3.33 kg/day with a dose of 13.93 g; and intake of acid detergent fiber (ADF) of 2.12 kg/day with a dose of 10.51 g. Digestibility coefficients showed a quadratic effect for doses of inclusion of calcium hydroxide. Dry matter digestibility was 72.88% with a dose of 11.65 g of calcium hydroxide, OM digestibility was 75.12% with 12.09 g; NDF digestibility was 58.67% with 12.11 g; ADF digestibility was 52.97% with 9.36 g and TDN digestibility was 71.42% with 10.92 g. There was no effect of the treatment for activities of rumination and total idle. Chewing time, number of bolus chewed and time for rumination of each bolli presented effect. Calcium hydroxide enabled greater intakes and digestibility of nutrients, especially from the cell wall, favoring the use of nutrients from the diet containing sugar cane, recommending a dose of 8-12 g.

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

This study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 μM cysteamine (C+C); 100 IU catalase (CAT) or 100 μM β-mercaptoethanol (β-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66-100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6-48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6-93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.

Cooled semen for fixed-time artificial insemination in beef cattle

This study evaluated the use of cooled semen in a fixed-time artificial insemination (FTAI) program compared with frozen-thawed semen to improve pregnancy rates in beef cattle. Ejaculates of three bulls were collected and divided into two treatments: (1) frozen-thawed semen and (2) cooled semen. Egg-yolk extender without glycerol was used for the cooled semen treatment. Straws (25×106 spermatozoa) were submitted to cooling for preservation at 5°C for 24h, after which FTAI was performed. Nelore cows (n=838) submitted to FTAI were randomly inseminated using frozen-thawed semen or cooled semen. There was a 20% increase in the pregnancy per AI (P AI-1) using cooled semen compared with frozen-thawed semen (59.9±4.7 vs 49.4±5.0%; P<0.005). There was no difference in P AI-1 among the bulls (P=0.40). The frozen-thawed semen had fewer functional spermatozoa than did the cooled semen when evaluated by sperm motility (61.7 vs 81.0%), slow thermoresistance test (41.7 vs 66.7%) and hypoosmotic swelling test (38.3 vs 53.7%; P<0.05). The percentage of sperm abnormalities did not differ between the freeze-thawing and cooling processes (18.6 vs 22.1%; P>0.05). Because there was less damage to spermatozoa and improvement in P AI-1, the use of cooled semen instead of frozen-thawed semen is an interesting approach to increase reproductive efficiency in cattle submitted to a FTAI protocol.

Effect of breed and corpus luteum on pregnancy rate of bovine embryo recipients

The objective of this study was to evaluate pregnancy rates of recipients of different breed groups (Nellore and crossbreed), as well as the effects of size and type of the corpus luteum (CL) on plasmatic concentrations of progesterone and pregnancy rates of embryo recipients. A total of 152 heifers were synchronized with progesterone implants and on the day of embryo transfer, previously obtained by superovulation and frozen in ethylene glycol, the diameter and type of the corpus luteum (cavitary and compact) was measured and blood was collected for progesterone measurement. The pregnancy rate was 44.1%, with a diameter of corpus luteum higher in recipients that became pregnant (2.03±0.41) compared with non-pregnant ones (1.86±0.34 cm). Plasmatic concentrations of progesterone did not differ between pregnant (1.50±1.05) and non-pregnant (1.31±0.91 ng/mL) animals. The type of corpus luteum did not influence the pregnancy rates. Only Angus and crossbred Marchigiana differ among themselves in pregnancy rates (33.3 and 59.2%, respectively). The pregnancy probability was affected only by CL diameter, but not by P4 plasmatic concentration. Selection of the corpus luteum size at the time of embryo transfer is an important factor to increase pregnancy rates in recipients, and compact and cavitary corpora lutea do not influence the pregnancy rates of bovine embryo recipients. Nellore recipients have pregnancy rates that are satisfactory and comparable to crossbred (Bos taurus × Bos indicus) recipients.

Follicular dynamics and production of oocytes in young Nellore heifers with energetic supplementation

To verify the effects of energy supplementation and fat on follicular dynamics, metabolic profile and the production of oocytes, 15 young heifers (Bos taurus indicus) with an average age of 14 months and with an initial weight of 256.35 kg were assigned to two treatments according to weight and follicular population as evaluated by ultrasonography: in T1 (1.0 × M), animals received 100% of the energy requirements for maintenance; in T2 (1.7 × M), animals received 170% of the energy requirements for maintenance, achieved by the addition of 200 g of Megalac®. After a period of adaptation to the diet, the treatments, blood collection and follicular aspirations were started using a randomized design. The dry matter intake and weight gain were lower in the 1.0 × M group than in the 1.7 × M group. No differences were found in the plasma progesterone concentrations, albumin, glucose, urea or gonadotropin (FSH and LH) levels between the groups. The mean concentrations of cholesterol were higher in the 1.7 × M group. The total number of small (<4 mm) and medium follicles (4-8 mm) was not altered by the treatments, but the number of small follicles increased on days 1 and 2 of the estrous cycle, with higher values found in the 1.7 × M group. The average of the oocytes also increased (9.50±2.1 and 12.5±4.4 for the 1.0 × M and 1.7 × M groups, respectively). The rapid increase in the amount of energy offered in the diet changes the amount of follicles and oocytes available for follicular aspiration (OPU) in young heifers without changing their metabolic profile.

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers

Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

Pregnancy rate in lactating Bos indicus cows subjected to fixed-time artificial insemination and treated with different follicular growth inducers

The objective of this experiment was to evaluate the effects of replacing eCG with FSH or FSH/LH on the diameter of ovulatory follicles (DOF) and the pregnancy rates (PR) of lactating Bos indicus cows subjected to a synchronization of ovulation protocol and fixed-time artificial insemination (FTAI) based on progesterone, estradiol benzoate (EB), PGF2α, and estradiol cypionate (EC). On day 0, primiparous and multiparous Nellore cows (n = 329) at 68 days postpartum were body condition scored (BCS) and received a P4 implant (PRIMER 1.0 g P4) plus 2 mg EB intramuscularly (IM). At day 8, the implant was removed, and 250 μg cloprostenol were injected IM along with 0.5 mg EC. At the time of implant removal, the animals were divided into one of four treatments: cows received (G1) 1 mL of saline solution IM (Control; n = 80); (G2) 300 IU eCG IM (ECG300; n = 84); (G3) 15 mg FSH IM (Folltropin; n = 82); and (G4) 30 IU FSH/LH IM (Pluset; n = 83); all cows were subjected to FTAI 48-54 h after implant removal (day 10). Transrectal ultrasonography was performed to measure the DOF to FTAI and to estimate the pregnancy rate on day 35. A statistical analysis examined the effects of the treatment, technician, semen, BCS, calf age and parity on PR to FTAI. There was no significant difference in PR and DOF among the treatment groups (G1, 41.3%, 13.1 mm; G2, 46.4%, 14.5 mm; G3, 46.3%, 14.2 mm; and G4, 48.2%, 14.3 mm), but PR was higher in multiparous than in primiparous cows. Commercial FSH solutions (Pluset or Folltropin) do not promote increases in PR or DOF of lactating Nellore cows and are equal to eCG in promoting growth in preovulatory follicles and elevating the PR in Nellore cows with a moderate BCS subjected to a progesterone-based FTAI protocol with EC.

Fontes de amido no concentrado de bovinos superprecoces de diferentes classes sexuais

The aim of this study was to evaluate two sources of starch in the concentrate, and three sex classes, on the consumption of nutrients, the performance and carcass characteristics of young bovines in feedlot. Thirty Beefalo-Nellore crossbred with an average initial body weight of 234.38±26.17kg and average age of nine months was used. All animals were maintained in individual bays. A completely randomized design with 2 x 3 factorial (two starch source and three sexual categories) was used. The experimental period lasted 224 days (32 weeks). In the initial phase (0-17 weeks) the weight gain (WG) and average daily gain (ADG) were 150.62 and 108.62kg and 1.27 and 0.91kg/day, respectively, for corn and sorghum. In the final phase (17-32 weeks) the feed efficiency of animals fed with maize was similar to sorghum, with an average of 10.66%. There was a significant source of starch for carcass weight, averaging 273.00 and 231.95kg respectively for the treatments with maize and sorghum. There was a sex class effect for WG, ADG and feed efficiency, with WG average equal to 98.17, 124.70 and 119.70kg, average daily gain of 0.93, 1.19 and 1.14kg/day and 8.66, 12.24 and 11.09% respectively for heifers, steers and young bulls. The subcutaneous fat thickness (SFT) showed an average of 10.50, 5.97 and 5.51mm for heifers, steers and intact males. In conclusion, the use of corn as a source of starch in the initial phase of the feed for young cattle is recommended. However, in the final stage, there is no distinction between sources of starch in the concentrate, which can be corn or sorghum. Females had higher EGS, while the males, castrated and intact, had higher feed efficiency in the final phase of confinement.

Ruminal parameters of bovines fed diets based on sugar cane with doses of calcium hydroxide

The objective of this study was to evaluate the administration of different doses of calcium hydroxide mixed with sugar cane fed to cows by the pH, ammonia nitrogen and volatile fatty acids concentration in ruminal content. Four cows with fistulated rumen were distributed in a Latin square (4 × 4) in split plot. The treatments involved adding doses of 0, 8, 16 and 24 g/kg of calcium hydroxide to sugar cane in natura. Samples of rumen fluid were collected and the pH and concentration of N-NH3 was determined before (time zero) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 hours after the feeding time. The concentration of volatile fatty acids at 0, 3, 6, 9 and 12 hours was also determined. The calcium hydroxide contributed to maintain the pH of rumen fluid close to neutral. The average concentrations of N-NH3 in the ruminal fluid was 20.59, 20.49, 17.28 and 18.22 mg/100 mL for samples with calcium hydroxide at 0, 8, 16, 24 g/kg, respectively. There was an effect of the addition of calcium hydroxide on the volatile fatty acids concentration from before feeding until 12 hours after feeding time. There was an effect on doses tested for the concentration of volatile fatty acids. The calcium hydroxide added to the sugar cane influences on ruminal parameters. The dose of approximately 10 g of calcium hydroxide per kg sugar cane in natura is recommended.