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The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.

The objective was to evaluate reproductive tract development (ovary and uterus) and onset of puberty in two lines of Nellore heifers (Bos indicus) selected for postweaning weight. A total of 123 heifers, including 46 from the control Nellore line (NeC) and 77 from the selection Nellore line (NeS) were used. Every 18 to 21 days from 12 to 24 months of age, average ovarian area (OVA), endometrial thickness (ETh), and diameter of the largest follicle in each ovary were evaluated (using transrectal ultrasonography), and body weight, hip height, and body condition score were measured. There were no differences between NeS and NeC heifers for ETh or OVA (P < 0.05). Genetic selection for higher postweaning weight had no negative influence on the onset of puberty, with 52% and 48% of NeC and NeS heifers, respectively, pubertal at 24 months of age (P = 0.49). Heifers that reached puberty at the end of the study were heavier (NeC, 296.9 vs. 276.7 kg; NeS, 343.5 vs. 327.9 kg; P < 0.01) and younger (NeC, 23.4 vs. 24.2 mo; NeS, 22.7 vs. 24.0 months; P < 0.01) than those that did not. Furthermore, heifers that were heavier at weaning reached puberty earlier. Pubertal heifers had a greater OVA (4.15 vs. 3.14 cm(2); P < 0.01) and ETh (12.15 vs. 9.93 mm; P < 0.01) than nonpubertal heifers. Taken together, OVA and ETh had positive effects (P < 0.01) on the onset of puberty and were suitable indicator traits of heifer sexual precocity in pasture management systems. However, selection for weight did not alter ovarian or endometrial development, or manifestation of puberty at 24 months of age. Among the growth traits studied, weaning weight and weight at puberty had significant positive effects on manifestation of first estrus.

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development. We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the same was detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos.

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 × 10 6 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 H 2 O 2 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 H 2 O 2. 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.

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