Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer's recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Desempenho de bezerros holandeses alimentados após o desaleitamento com silagem de grãos úmidos ou grãos secos de milho ou sorgo

Avaliou-se o desempenho de bezerros alimentados após desaleitamento e até o abate com silagem de grãos úmidos ou grãos secos de milho ou sorgo para a produção de vitelos de carne rosa. Trinta bezerros holandeses foram distribuídos em delineamento em blocos casualizados, com cinco blocos e seis tratamentos, e alimentados com seis rações concentradas com teores similares de proteína (18,5% PB) e energia (3,2 Mcal EM/kg de MS), formuladas com: milho seco moído (MM); silagem de grãos úmidos de milho (SGUM); sorgo seco com tanino moído, (SCTM); silagem de grãos úmidos inteiros de sorgo com tanino (SGUISCT); sorgo seco sem tanino moído (SSTM); e silagem de grãos úmidos inteiros de sorgo sem tanino (SGUISST). Os animais foram recriados em piquetes coletivos até atingirem o peso pré-estabelecido para o abate (170 ± 10 kg PC). Não houve efeito das rações concentradas sobre a altura de cernelha, a idade ao abate, os dias no experimento e os ganhos de peso diários e totais. Os custos por kg de ganho de peso das rações contendo sorgo foram mais baixos que o das rações formuladas com milho. Todos os alimentos avaliados podem ser usados em rações concentradas para bezerros após o aleitamento, pois não comprometem o desempenho e conferem resultados similares.

Desempenho de bezerros holandeses alimentados até o desaleitamento com silagem de grãos úmidos ou grãos secos de milho ou sorgo

Avaliou-se o desempenho de bezerros alimentados até o desaleitamento com silagem de grãos úmidos ou grãos secos de milho ou sorgo para posterior produção de vitelos de carne rosa. Trinta bezerros holandeses foram distribuídos em delineamento em blocos casualizados, com cinco blocos e seis tratamentos, e alimentados com seis rações concentradas com teores similares de proteína (22,5% PB) e de energia (3,2 Mcal EM/kg MS), formuladas com: milho seco moído (MM); silagem de grãos úmidos de milho (SGUM); sorgo seco com tanino moído (SCTM); silagem de grãos úmidos de sorgo com tanino (SGUSCT); sorgo seco sem tanino moído (SSTM); e silagem de grãos úmidos de sorgo sem tanino (SGUSST). Os animais receberam sucedâneo de leite até atingirem o peso pré-estabelecido para desaleitamento (60 ± 5 kg). Não houve efeito das rações concentradas sobre a altura de cernelha, a idade ao desaleitamento, os consumos diários de MS diários e totais das rações concentradas, os ganhos de peso diários e totais e a conversão alimentar. Os custos por kg de ganho de peso das rações concentradas contendo sorgo foram mais baixos que o das rações concentradas formuladas com milho. Todos os alimentos avaliados podem ser usados em rações concentradas para bezerros em fase de aleitamento, pois não comprometem o desempenho e conferem resultados similares.

Strategies to improve fertility in Bos indicus postpubertal heifers and nonlactating cows submitted to fixed-time artificial insemination

Two experiments were designed to evaluate strategies to increase fertility of Bos indicus postpubertal heifers and nonlactating cows submitted to a fixed-time artificial insemination (TAI) protocol consisting of an intravaginal device containing 1.9 g of progesterone (CIDR) insertion + estradiol benzoate on Day 0, CIDR withdrawal + estradiol cypionate on Day 9, and TAI on Day 11. In Experiment 1, heifers (n = 1153) received a new or an 18-d previously used CIDR and, on Day 9, prostaglandin F(2 alpha) (PGF(2 alpha)) + 0, 200, or 300 IU equine chorionic gonadotropin (eCG). Heifers treated with a new CIDR had greater (least squares means +/- SEM) serum concentration of progesterone on Day 9 (3.06 +/- 0.09 ng/mL vs. 2.53 +/- 0.09 ng/mL; P < 0.05) and a smaller follicle at TAI (11.61 +/- 0.11 nim vs. 12.05 +/- 0.12 mm; P < 0.05). Heifers with smaller follicles at TAI had lesser serum progesterone, concentrations on Day 18 and reduced rates of ovulation, conception, and pregnancy (P < 0.05). Treatment with eCG improved (P < 0.05) follicle diameter at TAI (11.50 +/- 0.10 mm, 11.90 +/- 0.11 mm, and 12.00 +/- 0.10 mm, for 0, 100, and 200 IU, respectively), serum progesterone concentration on Day 18 (2.77 +/- 0.11 ng/mL, 3.81 +/- 0.11 ng/mL, and 4.87 +/- 0.11 ng/mL), and rates of ovulation (83.8%, 88.5%, and 94.3%) and pregnancy (41.3%, 47.0%, and 46.7%). In Experiment 2, nonlactating Nelore cows (n = 702) received PGF(2 alpha) treatment on Days 7 or 9 and, on Day 9, 0 or 300 IU cCG. Cows receiving PGF(2 alpha) on Day 7 had lesser serum progesterone concentrations on Day 9 (3.05 +/- 0.21 ng/mL vs. 4.58 +/- 0.21 ng/mL; P < 0.05), a larger follicle at TAI (11.54 +/- 0.21 mm vs. 10.84 +/- 0.21 mm; P < 0.05), and improved (P < 0.05) rates of ovulation (85.4% vs. 77.0%), conception (60.9% vs. 47.2%), and pregnancy (52.0% vs. 36.4%). Treatment with eCG improved (P < 0.05) serum progesterone concentration on Day 18 (3.24 +/- 0.14 ng/mL vs. 4.55 +/- 0.14 ng/mL) and the rates of ovulation (72.4% vs. 90.0%) and pregnancy (37.5% vs. 50.8%). In conclusion, giving PGF(2 alpha) earlier in the protocol in nonlactating cows and eCG treatment in postpubertal heifers and nonlactating cows improved fertility in response to a TAI (progesterone + estradiol) protocol. (C) 2009 Elsevier B.V. All rights reserved.

Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows II: Strategies and factors affecting fertility

In Experiments 1, 2, and 3, we evaluated the effects of temporary weaning (TW), equine chorionic gonadotropin (eCG), and follicle-stimulating hormone (FSH) treatments on results of it fixed-time artificial insemination (TAI) protocol in postpartum Bos indicus cows. In Experiment 1, treatment with 400 IU eCG or with TW for 48 h consistently improved pregnancy rates (PRs) at TAI, but, in Experiment 2, FSH treatment was less effective than eCG or TW. In Experiment 3, the inclusion of eCG treatment in cows subjected to TW did not improve PRs. We concluded that TW or 400 IU eCG should be included in the TAI protocol in postpartum Bos indicus cows to enhance fertility. In Experiment 4, we used records front heifers and cows treated with the proposed protocol during the 2006-2007 (n = 27,195) and 2007-2008 (n = 36,838) breeding seasons from multiple locations in Brazil to evaluate factors potentially affecting PRs. Overall PR at TAI was 49.6% (31,786 of 64,033). Pregnancy rate differed (P < 0.01) among farm within location (results ranging between 26.8% and 68.0%; P < 0.01), cow group within farm, by breed (Bos indicus, 48.3% [26,123 of 54,145]; Bos taurus, 61.7% [3652 of 5922]; and crossbred Bos indicus x Bos taurus, 50.7% [2011 of 3966]), category (nulliparous, 39.6% [2095 of 52901] suckled primiparous, 45.2% [3924 of 8677]; suckled multiparous, 51.8% [24,245 of 46,767]; and nonsuckled multiparous, 46.1% [1522 of 3299]), body condition score at TAI (<= 2.5 43.0% [3409 of 7923]; 3.0, 49.6% [18,958 of 38,229]; and >3.5, 52.7% [9419 of 17,881]). Days postpartum at beginning, of protocol did not affect PR (30 to 60 d, 47.6% [4228 of 8881]; 61 to 90 d, 51.7% 116,325 to 31,5721; and 91 to 150 d, 50.8% [7616 to 14,991]; P > 0.1). Pregnancy rate was also consistently affected (P < 0.01) by sire (results ranging from 7.2% to 77.3%) and artificial insemination technician (results ranging from 15.1% to 81.8%). (C) 2009 Elsevier B.V. All rights reserved.

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AT of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with >= 70% morphologically normal sperm and 60% estimated progressive motility before cryopmservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). on Day 9 the CIDR was removed, 12.5 mg of PGF(2 alpha) and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day II (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS >= 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. on Day 7, all heifers received PGF(2 alpha) (12.5 mg). on Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI. (C) 2012 Elsevier B.V. All rights reserved.

Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows I: Basis for development of protocols

Five experiments were conducted on commercial farms in Brazil aiming to develop a fixed-time artificial insemination (TAI) protocol that achieved pregnancy rates between 40% and 55% in Bos indicus cows. These studies resulted in the development of the following protocol: insertion of all intravaginal device containing 1.9 g of progesterone (CIDR) plus 2.0 mg im estradiol benzoate on Day 0; 12.5 mg im dinoprost tromethamine on Day 7 in cycling cows or oil Day 9 in anestrous cows; CIDR withdrawal plus 0.5 mg im estradiol cypionate plus temporary calf removal on Day 9; TAI (48 h after CIDR withdrawal) plus reuniting of calves with their dams on Day 11. Reduced dose of prostaglandin F(2 alpha) (PGF(2 alpha): 12.5 mg im dinoprost tromethamine) effectively caused luteolysis. In cycling cows, fertility was greater when the treatment with PGF(2 alpha) was administered on Day 7 than oil Day 9, but in anestrous cows, no effects of time of the PGF(2 alpha) treatment were found. Estradiol cypionate effectively replaced estradiol benzoate or gonadotropin-releasing hormone as the ovulatory stimulus, reducing labor and cost. In this protocol, CIDR inserts were successfully used four times (9 d each use) with no detrimental effects on fertility. (C) 2009 Elsevier B.V. All rights reserved.

Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F-2 alpha

In Exp. 1, we evaluated the effects of 2 lengths of progesterone exposure [CIDR (controlled intravaginal drug release); 7 vs. 14 d] before a modified CO-Synch protocol [50.0-mu g injection of GnRH 6.5 d before a 25.0-mg injection of PGF(2 alpha) followed by another injection of GnRH and fixed-time AI (TAI) 2 d after PGF(2 alpha)], with or without temporary weaning (TW) before GnRH treatments, on fertility of suckled multiparous Bos indicus cows (n = 283) and on calf performance. Timed AI pregnancy rates for cows receiving 7 d CIDR + TW, 7 d CIDR, 14 d CIDR + TW, and 14 d CIDR were 53, 47, 46, and 41%, respectively (P > 0.10). Calves submitted to two 48-h TW 6 d apart had decreased mean BW at 240 d (187.9 +/- 2.7 vs. 195.5 +/- 2.7 kg; P < 0.05), but BW at 420 d was not affected by TW (240.1 +/- 5.1 kg). In Exp. 2, we evaluated the effect of no treatment and treatment with or without a CIDR insert between GnRH and PGF(2 alpha) treatments of a modified CO-Synch protocol on pregnancy rate to TAI, and throughout a 90-d breeding season in suckled multiparous Bos indicus cows (n = 453). The inclusion of a CIDR between first GnRH and PGF(2 alpha) treatments of a modified CO-Synch protocol did not improve pregnancy rate (29 and 33% for cows receiving CO-Synch + CIDR and CO-Synch protocol, respectively), and cycling cows had poorer TAI pregnancy rates than anestrous cows treated with either synchronization protocol (21.7 vs. 40.7%; P < 0.05). However, regardless of treatment with CIDR, cows submitted to TAI protocol had greater (P < 0.05) pregnancy rates at 30 (54.8 vs. 11.2%), 60 (72.1 vs. 38.8%), and 90 d (82.0 vs. 57.9%) of breeding season than untreated cows.

Effects of temperament on pregnancy rates to fixed-timed AI in Bos indicus beef cows

The objective of the present experiment was to assess the effects of temperament on pregnancy rates to fixed-time AI (FTAI) in Bos indicus beef cows. A total of 761 multiparous lactating Nelore cows, originated from 4 different commercial cow-calf ranches, were evaluated for BCS and temperament at the time of FTAI (day 0). Temperament was assessed by chute score and exit velocity. Further, individual exit score was calculated by dividing exit velocity results into quintiles and assigning cows with a score from 1 to 5 (exit score: 1 = slowest cows; 5 = fastest cow). Temperament scores were calculated by averaging cow chute score and exit score. Cows were also classified for temperament type according to temperament score (<= 3 = adequate temperament, >3 = excitable temperament). Pregnancy status was verified by detecting a viable conceptus with rectal ultrasonography approximately 40 days after FTAI. Chute score, exit velocity, and temperament score were not correlated to BCS (P>0.31). Hence, BCS did not differ (P=0.30) according to temperament type (4.13 vs. 4.09 for cows with excitable and adequate temperament, respectively; SEM = 0.070). Pregnancy rates to FTAI tended to be negatively affected by temperament score (P=0.08), whereas the probability of cows becoming pregnant to FTAI was negatively associated with temperament score (linear effect, P<0.01). Accordingly, pregnancy rates were reduced (P=0.05) in cows with excitable temperament compared to cows with adequate temperament (35.3 vs. 42.8% of pregnant cows/total cows, respectively; SEM = 2.85). Pregnancy rates to FTAI were not affected by chute score (P=0.25), although the probability of cows becoming pregnant to FTAI tended to be negatively associated with chute score (linear effect, P=0.07). Pregnancy rates to FTAI were negatively affected by exit score (P=0.05), and the probability of cows becoming pregnant to FTAI was negatively associated with exit score and with actual exit velocity (linear effects, P<0.01). Results from this experiment indicate that excitable temperament is detrimental to pregnancy rates of B. indicus cows assigned to an estrus synchronization + FTAI protocol. Published by Elsevier B.V.