Injectable progesterone in 13-month-old prepubertal Nellore heifers: effects on in vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of injectable progesterone administration before ovum pick-up (OPU) on in vitro embryo production (IVEP) and pregnancy rate in prepubertal Nellore heifers. Twenty-three Nellore females, 13.04 ± 0.8 months of age, were randomly assigned to two groups: control group (no progesterone application) and progesterone group (intramuscular administration of 1 mL [150 mg/mL] progesterone 7 days before OPU). The second OPU was performed after 28 days (crossover between groups). After OPU, oocytes were selected for IVEP and a sample was fixed in 4% paraformaldehyde. Viable oocytes were subjected to immunofluorescence staining for IGFBP2 and caspase-3. For each oocyte, one image was generated per channel (A555 and A488 filters) and analyzed with the ImageJ program for the quantification of fluorescence intensity (in pixels). The produced embryos were transferred to multiparous Nellore cows and pregnancy was diagnosed by ultrasound 30 days after transfer. Data were analyzed using the MIXED and GLIMMIX procedure of SAS®. Treatment with injectable progesterone had no effect on the number or size of the antral follicles of heifers. No differences between the control and progesterone groups were observed for total number of aspirated oocytes (P = 0.78), viable oocytes (P = 0.80), cleavage rate (P = 0.60), or blastocyst rate (P = 0.56). There were no differences in pixel intensity between the two groups, nor in the levels of IGFBP2 or caspase-3. However, a difference was observed in the pregnancy rate of embryos produced from heifers of the progesterone group (42.1%; 32/76) when compared to the rate of embryos produced from control heifers (28.4%; 23/81) (P = 0.02). The use of injectable progesterone before OPU in prepubertal Nellore heifers does not improve IVEP but increases the pregnancy rate after transfer.

Relationship between age, scrotal circumference, postweaning weight and semen quality in Nellore and Caracu bulls: a cross sectional study.

The aim of this study was to investigate the relationship between age, scrotal circumference, postweaning weight and semen quality in Nellore and Caracu bulls selected for postweaning weight. Data from the andrological evaluation of 836 bulls born between 2000 and 2019, including 583 Nellore animals (Bos indicus) and 253 Caracu animals (Bos taurus), were used. The bulls were divided into categories of age at the time of assessment: category 1 consisted of animals aged 20 to 23 months (22 ± 0.76 months, 518 ± 94.17 kg), category 2 consisted of animals aged 24 to 35 months (30 ± 4.42 months, 679 ± 137.19 kg), and category 3 consisted of animals ≥ 36 months (60 ± 14.12 months, 907 ± 161.73 kg). The statistical model included the effects of breed, age category, date of semen collection, and breed x age category interaction. Heritability estimates for scrotal circumference at 13 months of age (SC1year) and semen quality traits were obtained for the sample of Nellore animals. Most semen quality traits improved with increasing age in both Nellore and Caracu animals. High heritability was observed for SC1year (0.45), while sperm motility, vigor, turbulence, and major, minor and total sperm defects exhibited low heritability (0.11, 0.019, 0.047, 0.017, 0.017 and 0.019, respectively). Spearman correlations of breeding values for postweaning weight (W378) and SC1year with the semen quality traits were low. Nellore and Caracu bulls have similar semen quality that improves with increasing age. In the Nellore breed, the heritability of SC is high, while semen quality traits exhibit low heritability. Selection for higher postweaning weight does not phenotypically affect the semen quality of bulls at breeding age.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Use of auxiliary techniques for assessing the reproductive capacity of natural service Nellore bulls.

The aim of this study was to investigate whether parameters obtained by computer-assisted sperm analysis (CASA), infrared thermography (IRT), and testicular B-mode and Doppler ultrasound can be used as indicators of fertility in natural service Nellore bulls. Twenty-nine Nellore bulls were evaluated on days 66 (-D66), 38 (-D38), and 10 (-D10) before the beginning of natural service mating. In all assessments, semen samples were collected and sperm kinetics were evaluated by CASA, thermographic imaging of the scrotal and ocular region, testicular B-mode ultrasound, and Doppler ultrasound of the spermatic cord. At the end of natural service, the bulls were classified based on the pregnancy rate of individual batches into low fertility (pregnancy rate 66.57 ± 0.62 %), medium fertility (76.47 ± 0.51 %), and high fertility (84.80 ± 0.60 %) groups. No difference in the IRT parameters was observed between groups. High fertility animals exhibited higher average path velocity (VAP = 110.98 µm/s), straight line velocity (VSL = 87.05 µm/s), curvilinear velocity (VCL = 190.78 µm/s), pulsatility index (PI = 0.93), and resistive index (RI = 0.57) than low fertility animals (VAP = 100.02 µm/s; VSL = 79.84 µm/s; VCL = 173.22 µm/s; PI = 0.69; RI = 0.48). Positive correlations were observed between pregnancy rate and VSL (0.21), PI (0.28) and RI (0.32). In conclusion, IRT does not provide fertility indicators in Nellore bulls. The VAP, VSL and VCL obtained by CASA and PI and RI obtained by Doppler ultrasound can be used as indicators of fertility in Nellore bulls.

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency.

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.

Effect of Nellore (Bos indicus) donor age on in-vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of age of Nellore (Bos indicus) donors on the efficiency of in vitro embryo production (IVEP) and pregnancy rate. Thirty-six donors, including 11 female calves (13 ± 0.61 months), 17 prepubertal heifers (25 ± 0.78 months) and 8 cows (83 ± 28 months), were submitted to 3 procedures of ovum pickup (OPU) on random days of the estrous cycle at intervals of 21 days. Caspase-3 and IGFBP2 were quantified in oocytes and blastocysts for the evaluation of oocyte and embryo quality. The produced embryos were vitrified (n = 445) and transferred to synchronized recipients. Cows produced a larger number of follicles (cows: 54.5 ± 6.2; calves: 20.0 ± 0.57; prepubertal heifers: 20.8 ± 0.46), total oocytes (cows: 45.97 ± 7.22; calves: 28.93 ± 6.14; prepubertal heifers: 27.21 ± 4.94) and cleaved oocytes (cows: 21.14 ± 4.22; calves: 13.09 ± 3.72; prepubertal heifers: 12.4 ± 3.19). The cleavage rate was similar between age categories; however, cows tended (p < 0.07) to produce a larger number of blastocysts (9.74 ± 2.26) per OPU than calves (5.57 ± 1.99) and prepubertal heifers tended to have a higher blastocyst yield (35.4%) than calves (27.1%) (p < .07). The expression levels of IGFBP2 and caspase-3 were higher in oocytes derived from calves compared to the other two categories. The pregnancy rate was higher in calves (43.1%) and cows (40.4%) than in prepubertal heifers (33.8%) (p = .03). Despite the larger numbers of follicles and viable oocytes in cows, the blastocyst production results and pregnancy rates obtained indicate that the use of young females as oocyte donors in IVEP is feasible and may contribute to reduce the generation interval.

Effects of paternal diet and antioxidant addition to the semen extender on bovine semen characteristics and on the phenotype of the resulting embryo.

The aim of this study was to examine the effects of long-term dietary supplementation of young Nellore bulls with rumen-protected polyunsaturated fatty acids (PUFAs) and of the inclusion of catalase in the semen extender on semen quality, in vitro sperm fertilizing ability, and intracytoplasmic lipid content in the resulting embryos. Twelve Nellore bulls were supplemented with rumen-protected PUFAs or with a basal diet from 14 to 24 months of age. The semen was collected at the end of supplementation. For cryopreservation, the ejaculate was divided into two equal volumes and catalase was added to the extender in one of the fractions. Thus, the experimental design consisted of a 2 × 2 factorial scheme with two diets (control and PUFA) and two extenders (Cat+ and Cat-). Total motility and the percentage of rapid cells in fresh semen were negatively affected by dietary supplementation with PUFAs (P < 0.05), but these effects did not persist after freezing. The frozen/thawed semen of animals fed PUFAs exhibited an increase in the percentages of damaged plasma and acrosomal membranes, as well as an increase in the proportion of lipids ions at m/z 578 and m/z 757 detected by MALDI-MS. Nevertheless, there was no effect of the treatments on in vitro embryo development. However, embryos derived from bulls supplemented with PUFAs exhibited higher lipid accumulation compared to control (P < 0.05). In conclusion, PUFA supplementation promoted worsening of semen quality without affecting the in vitro sperm fertilizing ability; however, the paternal diet affected the intracytoplasmic lipid content in the resulting embryos.

Sperm viability, serological, molecular, and modified seminal plasma agglutination tests in the diagnosis of Leptospira in the semen and serum of bovine bulls.

The present study investigated the serum microscopic agglutination test (MAT) among 203 bovine bulls with reproduction by natural means, without apparent signs of orchitis or inflammation of accessory reproductive glands. Simultaneously, the semen of all bulls was subjected to sperm viability analysis and PCR based on the 16S rRNA gene. PCR-positive results of semen samples were confirmed by sequencing. A modified seminal plasma agglutination (MSPA) test, replacing the blood serum of all bulls in the MAT with seminal plasma was performed as well. Eight (8/203 = 3.9%) semen samples from bulls were considered nonviable (necrospermia and azoospermia) without relation to the PCR diagnosis. No agglutinin titers were identified in MSPA test. A high frequency (132/203 = 65%) of leptospiral agglutinin titers was identified in the MAT, particularly for the Sejroe serogroup (Hardjo CTG, 100/203 = 49.3%; Wolffi 74/203 = 36.4%; Guaricura 72/203 = 35.5%; and Hardjoprajitno 56/203 = 27.6%). Three (3/203 = 1.5%) semen samples of bulls were positive in the PCR, but these results were not confirmed by sequencing. The high frequency of serovars from the Sejroe serogroup typically adapted to bovines indicates the need for measures for the prophylaxis/control of the pathogen on the sampled farms. Discrepancies among the MAT, sperm viability, and molecular detection of leptospires in semen highlight the need for a combination of methods to diagnose leptospirosis in bovine bulls. To our knowledge, modified seminal plasma agglutination is described for the first time here to investigate anti-Leptospira antibodies produced locally in the genital tract in the diagnosis of bovine leptospirosis among bulls that reproduce by natural means.

Ultrasonographic characteristics of the testes, epididymis and accessory sex glands and arterial spectral indices in peri- and post-pubertal Nelore and Caracu bulls.

Ultrasonography can provide information about the integrity of organs; however, rarely is applied to the reproductive organ evaluation of bulls. The objective of the present study was to characterize and compare values for variables and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as spectral Doppler indices of the testicular and internal iliac arteries, between peri- and post-pubertal Nelore and Caracu bulls. Nelore (n = 203) and Caracu (n = 79) bulls were assigned by age class: peri-pubertal (12-15 months) and post-pubertal (> 22 months). Data were analyzed using SAS's PROC MIXED procedure (P < 0.05). The biometric variables of the testes and cauda epididymis differed between peri- and post-pubertal Nelore and Caracu bulls. There was a difference between breeds for the vesicular glands, ampulla of vas deferens, disseminate portion of the prostate, and craniocaudal dimension of the bulbourethral glands. Echogenicity of the testicular parenchyma differed between breeds and age classes. The pulsatility and resistive indices of the testicular arteries differed between Nelore and Caracu bulls. The biometric and ultrasonographic characteristics of the testes, epididymis and accessory sex glands, as well as of the arterial indices in bulls are affected by genetic group and age class, and when assessed there is useful information regarding the progression of sexual maturation.

Sexual maturity and fertility-related measures in young Nellore bulls receiving long-term dietary supplementation with rumen-protected polyunsaturated fatty acids.

The objective of this study was to evaluate the effects of long-term supplementation with rumen-protected fatty acids (FA) on growth and reproductive parameters of young Nellore bulls in a grazing regime. Forty-eight young bulls were distributed into two groups: FA (supplemented with rumen-protected polyunsaturated FA); and control (control fat-free supplement). The animals were supplemented from 14.3 to 24.6 months of age and growth and reproductive parameters were evaluated at 28-day intervals. The semen was cryopreserved in the last collection and fresh and post-thaw semen samples were evaluated. Feeding FA did not affect (P > 0.05) growth, reproductive parameters (scrotal circumference, sperm concentration per mL of ejaculate, percentage of sperm defects, sperm quality and fertility in vitro), or testicular ultrasonographic characteristics. However, thawed semen from bulls fed FA exhibited better quality (P < 0.05) than control semen for the following parameters evaluated by computer-assisted sperm analysis: average path velocity [µm/s: 90.48 vs. 79.66 post-thaw and 74.81 vs. 72.80 post-rapid thermoresistance test (TRT)], straight-line velocity (µm/s: 72.37 vs. 65.20 post-thaw and 64.96 vs. 63.25 post-TRT), and curvilinear velocity (µm/s: 148.44 vs. 131.31 post-thaw and 115.68 vs. 113.35 post-TRT). In addition, feeding FA increased peripheral concentrations of testosterone, leptin, total cholesterol and high-density lipoprotein. In conclusion, the increase in testosterone concentrations in bulls fed FA was not related to variations in growth parameters and sexual maturity. In addition, post-thawing sperm velocities were enhanced by diet, however, such increases were not related to better in vitro embryo production rates.

Evaluation of cooling and freezing systems of bovine semen.

Semen cryopreservation comprises different steps, among them are the cooling and freezing rates which significantly influence the quality of thawed sperm. Different systems with variable freezing rates are used for freezing bull semen in the field, with a consequence of variable success rates. The objective of this study was to compare different systems for freezing bull semen in the field. Five cooling methods of semen and two methods for the subsequent freezing phase (5 × 2 factorial scheme) were used. Two to four ejaculates were collected from 12 bulls with an electroejaculator. The ejaculates were diluted in BotuBov® to a concentration of 50 × 106 spermatozoa/mL in 0.5-mL straws. After dilution, the straws were cooled to 5 °C in five cooling systems: TK 4000® at a cooling rate of -0.25 °C/min (R1); TK 4000® at a rate of -0.5 °C/min (R2); Minitube® refrigerator at a rate of -2.8 °C/min (R3); Botutainer® at a rate of -0.65 °C (R4), and domestic refrigerator at a rate of -2.0 °C/min (R5). After stabilization at 5 °C for 4 h, these straws were then submitted to two freezing systems: TK 4000® at a freezing rate of -15 °C/min (C1) and Styrofoam box with liquid nitrogen at a rate of -19 °C/min (C2). Sperm kinetics were evaluated by computer-assisted sperm analysis at four time points: in fresh semen, after cooling, post-thawing, and after the rapid thermal resistance test (TRT). In addition, plasma and acrosomal membrane integrity, mitochondrial potential and intracellular H2O2 were analyzed after thawing by flow cytometry. The R1, R2 and R4 cooling systems were the most efficient in preserving sperm viability, membrane integrity and intracellular H2O2. Samples frozen in the C1 system exhibited better post-thaw and post-TRT kinetics than C2 samples. In conclusion, slower cooling curves in conjunction with a constant freezing rate obtained with the programmable unit were more efficient for freezing bull semen in the field.

Corpus luteum dynamics after ovulation induction with or without previous exposure to progesterone in prepubertal Nellore heifers.

The objectives of this study were 1) to monitor corpus luteum (CL) dynamics after two different protocols of ovulation induction in prepubertal Nellore heifers, and 2) to determine differences in luteal function. Fifty-seven heifers (weight 289.61 ± 32.28 kg, BCS 5.66 ± 0.65, age 17.47 ± 0.81 months) were divided into two groups: GP4+GnRH received a progesterone (P4) device of 3rd use for 10 days, followed by the administration of 0.02 mg buserelin acetate (GnRH) 48 h after removal of the device, and GGnRH received only GnRH. The CLs formed were monitored by ultrasonography every 2 days until their functional regression (decrease in the color Doppler signal and serum P4 concentration < 1 ng/mL), determining their diameter and area, numerical pixel value (NPV), pixel heterogeneity, and vascularization percentage. The peak systolic velocity, end diastolic velocity, resistivity index and pulsatility index (PI) of the ovarian artery and serum P4 concentration were also measured. A lifespan of the CL of more than 16 days was classified as normal-function and of less than 16 days as premature regression. The variables were compared between treatments, CL categories (normal-functional, prematurely regressed or non-functional), days of evaluation, and their interactions using the MIXED procedure of the SAS program (p ≤ 0.05). Three animals of each group (6/57 = 11%) did not respond to treatment, corresponding to an ovulation rate of 89%. There was a higher percentage of normal-function CLs in GP4+GnRH (81%) and a higher percentage of non-functional CLs in GGnRH (52%; P4 concentration < 1 ng/mL in all assessments). Normal-function CLs exhibited a greater area, vascularization percentage and P4 concentration than prematurely regressed and non-functional CLs. Lower diameter, area, NPV and P4 concentration were observed for non-functional CLs, but there was no difference in vascularization percentage compared to prematurely regressed CLs. Progesterone concentration was efficient in diagnosing CL function and was positively correlated with CL area (r = 0.62; p < 0.001) and vascularization percentage (r = 0.38; p < 0.001). Diameter and PI were important for the early diagnosis of non-functional and prematurely regressed CLs, respectively. In conclusion, luteal function differed for the first CL that develops after ovulation induction in prepubertal heifers. Ultrasonographic parameters (diameter, area, NPV, vascularization percentage, and PI) can be used to predict CL function.

Digestion and metabolism of low and high residual feed intake Nellore bulls.

Understanding the reasons why animals of similar performances have different feed requirements is important to increase profits for cattle producers and to decrease the environmental footprint of beef cattle production. This study was carried out aiming to identify the associations between residual feed intake (RFI) and animal performance, nutrient digestibility, and blood metabolites related to energy balance of young Nellore bulls during the finishing period. Animals previously classified as low (n = 13) and high RFI (n = 12), with average initial body weight of 398 kg and age of 503 days were used. Cattle were fed a high energy diet and were slaughtered when rib fat thickness measured by ultrasound between the 12th and 13th ribs reached the minimum of 4 mm. A completely randomized design was adopted, being data analyzed with a mixed model that included the random effect of slaughter group, the fixed effect of RFI class, and linear effect of the covariate feedlot time. No differences were found (p > 0.10) between RFI classes for performance, dry matter, and nutrients intake. However, dry (p = 0.0911) and organic matter (p = 0.0876) digestibility tended to be lower, and digestibility of neutral detergent fiber corrected for ash and protein (p = 0.0017), and total digestible nutrients (p = 0.0657) were lower for high RFI animals, indicating lesser capacity of food utilization. Difference between low and high RFI animals was also found for blood cortisol at the end of the trial (p = 0.0044), having low RFI animals lower cortisol concentrations. Differences in the ability to digest food can affect the efficiency of transforming feed into meat by Nellore cattle.

Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility.

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 106 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

Assessment of different in vitro sperm challenges and in vivo fertility of bovine semen batches / Avaliação de diferentes desafios espermáticos in vitro e da fertilidade in vivo de partidas de sêmen bovino

The aim of this work was to submit sperm cells to different laboratory challenges and to compare in vitro results with in vivo semen fertility. Four different batches from the same Brangus bull were used in a timed-AI program of 332 Brangus cows. Each batch (B) was submitted to the following procedure: semen sample was thawed at 36°C for 30 seconds (control). Sperm motility parameters, plasma membrane integrity, sperm morphology, and concentration were assessed. Then, an aliquot of thawed sample was incubated in a water bath at 45°C for 40 min (thermal challenge group; TCG) and another aliquot was centrifuged at 500 xg (Percoll gradient 45%/90%) for 15 min (centrifugation challenge group; CCG). Centrifuged semen was also submitted to another thermal challenge, being incubated (water bath) at 45°C for 40 min (centrifugation + thermal challenge group; CTCG). At the end of each challenge (CCG, TCG, and CTCG), the same laboratory tests used for control group were repeated. The following conception rates (CR) were observed for each batch: B1 = 48.9% (44/90); B2 = 44.2% (23/52); B3 = 55.5% (40/72); B4 = 43.2% (51/118); (p < 0.10). In the lab, B3 presented higher (p ≤ 0.05) progressive motility (PM) than B4 after thawing (control group) and after all sperm challenges (TCG, CCG, and CTCG). However, despite B3 and B4 having demonstrated a similar percentage of plasma membrane integrity (PMI) to the control group (B3 = 66.7 ± 1.3 and B4 = 65.2 ± 3.3), B3 demonstrated higher (P ≤ 0.05) percentage of PMI (37.2 ± 2.5) than B4 (26.7 ± 3.3) after passing through the most stressing in vitro challenge (CTCG). The semen batch presenting the highest resistance to in vitro challenges was the one that presented a trend for higher in vivo fertility, suggesting that submitting semen samples to laboratory challenges may be an interesting alternative for selecting batches with greater field fertility.(AU)

O objetivo deste estudo foi estressar células espermáticas em diferentes desafios laboratoriais e comparar os resultados in vitro com a fertilidade in vivo do sêmen. Quatro partidas de um mesmo touro Brangus foram utilizadas em um programa de IATF de 332 vacas Brangus. Cada partida foi submetida ao seguinte procedimento: a amostra de sêmen foi descongelada a 36°C por 30 segundos (grupo controle). Foram avaliados parâmetros de motilidade espermática (CASA), integridade da membrana plasmática (PMI), morfologia e concentração espermática. Em seguida, uma alíquota da amostra descongelada foi incubada em banho-maria a 45°C durante 40 minutos (grupo de desafio térmico, TCG) e outra alíquota foi centrifugada a 500 xg (gradiente de Percoll 45%/90%) durante 15 min (grupo desafio de centrifugação, CCG). Uma aliquota do sêmen centrifugado foi ainda submetida ao desafio térmico, sendo incubado a 45°C durante 40 min (grupo de desafio térmico + centrifugação, CTCG). No final de cada desafio (CCG, TCG e CTCG), os mesmos testes laboratoriais utilizados para o grupo de controle foram realizados. A seguinte taxa de concepção (CR) foi observada para cada partida (B): B1 = 48,9% (44/90), B2 = 44,2% (23/52), B3 = 55,5% (40/72) e B4 = 43,2% (51/118); (P < 0,10). No laboratório, B3 apresentou maior (P ≤ 0,05) motilidade progressiva (PM) do que B4 logo após o descongelamento (grupo controle) e após todos os desafios laboratoriais (TCG, CCG e CTCG). Porém, apesar de B3 e B4 demonstrarem similar porcentagem de PMI no grupo controle (B3 = 66,7 ± 1,3 e B4 = 65,2 ± 3,3), B3 apresentou maior (P ≤ 0,05) PMI (37,2 ± 2,5%) do que B4 (26,7 ± 3,3%) após passar pelo maior desafio laboratorial (CTCG). A partida seminal que in vitro apresentou maior resistência aos desafios laboratoriais foi a mesma que apresentou tendência para maior fertilidade in vivo. Assim, sugere-se que submeter amostras seminais a desafios laboratoriais pode ser uma alternativa interessante para selecionar partidas com maior fertilidade a campo.(AU)

Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.

Assessment of in vitro sperm characteristics and their importance in the prediction of conception rate in a bovine timed-AI program.

The aims of this study were to assess in vivo fertility and in vitro sperm characteristics of different sires and to identify sperm variables important for the prediction of conception rate. Multiparous Nelore cows (n = 191) from a commercial farm underwent the same timed artificial insemination (timed-AI) protocol. Three batches of frozen semen from three Angus bulls were used (n = 9). A routine semen thawing protocol was performed in the laboratory to mimic field conditions. The following in vitro sperm analyses were performed: Computer Assisted Semen Analysis (CASA), Thermal Resistance Test (TRT), Hyposmotic Swelling Test (HOST), assessment of plasma and acrosomal membrane integrity, assessment of sperm plasma membrane stability and of lipid peroxidation by flow cytometry and assessment of sperm morphometry and chromatin structure by Toluidine Blue staining. For statistical analyses, Partial Least Squares (PLS) regression was used to explore the importance of various sperm variables in the prediction of conception rate. The following in vitro sperm variables were determined to be important predictors of conception rate: total motility (TM), progressive motility (PM), TM after 2 h of thermal incubation (TM_2 h), PM after 2 h of thermal incubation (PM_2 h), Beat Cross Frequency after 2 h of thermal incubation (BCF_2 h), percentage of rapidly moving cells after 2 h of thermal incubation (RAP_2 h), intact plasma membrane evaluated by HOST, intact plasma and acrosomal membranes evaluated by flow cytometry, intact plasma membrane suffering lipid peroxidation, major defects, total defects, morphometric width/length ratio, Fourier_0 and Fourier_2 and Chromatin Heterogeneity. We concluded that PLS regression is a suitable statistical method to identify in vitro sperm characteristics that have an important relationship with in vivo bull fertility.

Chromatin modifying agents in the in vitro production of bovine embryos.

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation.

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.