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Contents The time at which follicles acquire LHR in bovine granulosa cells is the subject of some controversy among researchers. The main objective of the present study was to assess the mRNA expression of LHR and LRBP ( mRNA protein binding), a post-transcriptional suppressor of LHR mRNA expression, in granulosa cells from the two largest follicles around the expected time of follicle deviation in Nelore heifers. First, the interval between ovulation and follicle deviation in 20 Nelore heifers was determined (2.3 ± 0.2 days after ovulation). Ovulation was hormonally synchronized, and then, heifers were slaughtered on days 2, 2.5 and 3 after ovulation (before, during and after, respectively, the expected time of follicle deviation), and granulosa cells from the two largest follicles were collected. The m RNA abundance of an LHR fragment common to all isoforms (total LHR) and LRBP was assessed by real-time RT- PCR, and LHR alternative transcripts were assessed by semiquantitative RT- PCR followed by electrophoresis. LHR m RNA expression was not detected before the expected time of deviation. Total LHR m RNA abundance was greater in the largest follicle and increased from day 2.5 to 3. In contrast, LRBP mRNA was detected starting on day 2 and was more expressed in the second largest follicle on days 2.5 and 3. The present data suggest that the expression of LHR mRNA in bovine granulosa cells is established after follicle deviation and that the lower abundance of LRBP m RNA after the expected time of deviation may contribute to greater expression of LHR in the bovine dominant follicle. [ABSTRACT FROM AUTHOR]

Contents Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes ( COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes. [ABSTRACT FROM AUTHOR]

Contents [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O 2 - ) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O 2 - and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Considering the high temperatures that the tropical climate provides to most of Brazil and the effects of thermal stress on reproductive processes, the objective of the present study was to analyze, in the warmer months of 2016, conception rates of Nelore bovine embryos in Acre state. For this purpose, oocytes were aspirated (ultrasound-guided follicular aspiration), matured, fertilized with Nelore bull semen, cultured for 6 days, and then the embryos were transferred to crossbred recipients. Pregnancy diagnosis was performed 30 and 60 days after embryo transfer. Meteorological data were obtained at www.inmet.gov.br to generate temperature-humidity index (THI). The data from the conception rates and periods of the year were submitted to the chi-square test at 5% probability to verify independence. Regression analysis was used to verify the relationship between THI and gestation rate. There was a strong relationship between conception rates and THI values, verified by an increase in conception rates as THI values were reduced and a decrease when THI reached the highest value. Our findings demonstrated a negative effect of heat stress in conception rates of crossbred cows in northern Brazil.

Studies have shown that the use of equine chorionic gonadotropin (eCG), which binds both follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors, could modify the female reproductive tract. We, thus, aimed to quantify the messenger RNA (mRNA) abundance of genes related to cumulus-oocyte complexes (COCs) and embryo quality in Nelore cows (Bos taurus indicus) submitted to ovarian superstimulation using only FSH (FSH group; n = 10) or replacement of the last two doses of FSH by eCG (FSH/eCG group; n = 10). All animals were slaughtered and the ovarian antral follicles from both groups (10-14 mm in diameter) were aspirated for cumulus, oocyte and in vitro embryo production gene expression analysis. The relative mRNA abundance of 96 genes related to COCs development and embryo quality was measured by RT-qPCR. We found that oocytes are more affected by eCG use and that 35 genes involved in lipid metabolism, oxidative stress, transcriptional control, and cellular development were upregulated in the FSH/eCG group. In blastocysts, lipid metabolism seems to be the main pathway regulated by eCG use. We suggest that these multiple effects could be due to the ability of eCG to bind LHR and FSHR, which could activate multiple signal transduction pathways in the superstimulated ovary, further impacting the transcriptional profile of COCs and blastocysts., (© 2019 Wiley Periodicals, Inc.)

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells., (Copyright © 2017. Published by Elsevier Inc.)

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0 kV/cm) and time (20, 25, 50 and 60 μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75 kV/cm for 60 μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows., (Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Many peptides are responsible for the coordination of muscle contraction, secretion and ciliary beating of the oviduct epithelium to allow the transport of gametes and embryos, including vascular endothelial growth factors (VEGF), prostaglandins (PGs), endotelin-1 (ET-1) and angiotensin II (Ang II). The effect of reproductive biotechnologies used to improve embryo yield on oviduct gene expression is poorly understood. Thus, the aim of the present study was to evaluate the effect of ovarian superstimulation on the mRNA expression of the genes encoding the major peptides involved in oviduct contraction in bovine. Therefore, Nelore cows were submitted to P-36 (n=5) or P-36/eCG (n=5) ovarian superstimulatory protocols and a control group of cows was not submitted to any superstimulatory protocol (n=5). The relative expression of VEGF (VEGF, Flk1, Flt1), Ang II (AGTR2, ACE1), ET1 (ET1, ECE1) and PG pathway members (PGES, EP2, EP4, COX1, COX2) was analyzed using real time RT-PCR in each of oviduct segment (infundibulum, ampulla and isthmus). All target genes were expressed in the three segments of the bovine oviduct; however, specific genes were regulated by ovarian superstimulation: EP2 and EP4 receptors mRNA was affected by P-36/eCG protocol, in the ampulla and infundibulum, respectively; and AGTR2 mRNA was up-regulated by both the P-36/eCG and P-36 protocols in the isthmus. The upregulation of EP2, EP4 and AGTR2 expression in the superstimulated cows suggests a suitable effect of FSH and eCG on bovine oviduct physiology, coordinating the contraction in Nelore cows., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The LH plays a key role in controlling physiological processes in the ovary acting via LH receptor (LHR). In general, the effects of LHR on the regulation of granulosa cell differentiation are mediated mainly via the Gs-protein/adenylyl cyclase/cAMP system; however, the LHR activation could also induce phospholipase C (PLC)/inositol trisphosphate (IP3) via Gq/11 system. Additionally, the expression of G-proteins (GNAS, GNAQ, and GNA11) and PLC β has been showed in bovine antral follicle, concomitant with an increase in LHR expression. To gain insight into the effects of superstimulation with FSH (P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the mRNA expression of proteins involved in LHR signaling in bovine granulosa cells, Nelore cows (Bos indicus) were treated with two superstimulatory protocols: P-36 protocol or P-36/eCG protocol (replacement of the FSH by eCG administration on the last day of treatment). Nonsuperstimulated cows were only submitted to estrous synchronization without ovarian superstimulation. The granulosa cells were harvested from follicles and mRNA abundance of GNAS, GNAQ, GNA11, PLCB1, PLCB, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, ADCY8, and ADCY9) was measured by real-time reserve transcription followed by polymerase chain reaction. No differences on mRNA abundance of target genes were observed in granulosa cells of cows submitted to P-36 protocol compared with control group. However, the cows submitted to P-36/eCG protocol showed upregulation on the mRNA abundance of target genes (except ADCY8) in granulosa cells. Although the P-36 protocol did not regulate mRNA expression of the proteins involved in the signaling mechanisms of the cAMP and IP3 systems, the constant presence of GNAS, GNAQ, GNA11, PLCB1, PLCB3, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, and ADCY9) mRNA and the upregulation of these genes in granulosa cells from cows submitted to P-36/eCG protocol reinforce the participation of Gq/11/PLC/IP3 signaling as well as Gs-protein/adenylyl cyclase/cAMP system on LHR pathways during bovine granulosa cell differentiation submitted to superstimulatory treatments., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The insulin-like growth factor (IGF) system is related to quality of oocytes and embryos. The aim of this study was to investigate the mRNA levels of IGF1 and IGF2 and their receptors, IGFR1 and IGFR2, as well as IGFBP2, IGFBP4, and PAPP-A in oocytes from Nelore compared to Holstein cows. Pools of oocytes (20 oocytes/pool) from Nelore (n=8 pools) and Holstein (n=4 pools) were obtained via ovum pick-up (OPU, 10 sessions) and cumulus cells and zona pellucida were removed. The pools were submitted to total RNA extraction. Expression of members of the IGF system was assessed by real time RT-PCR. The mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4 was significantly higher (P<0.01) in oocytes from Holstein whereas the expression of PAPP-A was significantly higher (P<0.05) in oocytes from Nelore cows. The high PAPP-A expression and the low expression of IGFBP2 and IGFBP4 are associated with more efficient degradation of IGFBPs, which results in greater bioavailability of IGF in Nelore oocytes when compared to the Holstein., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The objective was to evaluate when the LH reserve was re-established in postpartum Nellore (Bos indicus) cows by evaluating the response of the hypothalamic-pituitary axis responsiveness to exogenous GnRH or estradiol benzoate (EB). Additionally, we tested the influence of dietary supplementation (SUPL) and calf removal (CR) on the duration of postpartum anestrus. Ninety multiparous lactating Nellore cows were randomly assigned to eight groups. The EB and GnRH groups received 1.0 mg EB (N = 7), and 50 μg lecireline (N = 16), respectively. Additional cows were given the same hormones, and subjected to either nutritional supplementation (EB-SUPL, N = 9; GnRH-SUPL, N = 16), or calf removal at 72 hours after calving (EB-CR, N = 4; GnRH-CR, N = 13). The remaining two groups were the LH (12.5 mg, N = 14) and control groups (saline, N = 11). Hormones were administered weekly from 7 (±5) days postpartum to first ovulation (detection of a CL during a weekly ultrasonographic examination). Blood samples were collected just before and 2 hours (GnRH, LH, and control groups) or 18 hours (EB groups) after hormone or saline (control) administration. Ovulation occurred as early as 15 days postpartum in the GnRH group. The mean ± SEM intervals (days) from calving to first ovulation were EB, 87.7 ± 4.2; EB-CR, 20.3 ± 1.2; EB-SUPL, 60.3 ± 3.2; GnRH, 40.4 ± 2.1; GnRH-CR, 21.0 ± 1.1; GnRH-SUPL, 26.4 ± 1.1; LH, 35.6 ± 1.1; and control, 60.9 ± 2.1. We concluded that there was sufficient LH in the pituitary gland (of Nellore cows) from the second week postpartum to induce ovulation in response to exogenous GnRH. Additionally, calf removal and nutritional supplementation reduced, by 2 to 4 weeks, the interval from calving to an LH increase and ovulation induced by GnRH or EB., (Copyright © 2013. Published by Elsevier Inc.)

High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P<0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P<0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

The aim of this study was to evaluate the effect of follicular fluid collected from bovine dominant follicles (bFF) on the in vitro development of goat preantral follicles and determine the best time to add this supplement to the culture medium. The preantral follicles were isolated and randomly distributed into four treatments in absence (control) or presence of 10% of bFF added on Days 0 (FF0-18), 6 (FF6-18) or 12 (FF12-18) of culture onwards. After 18 days, follicular development was assessed based on follicular survival, antral cavity formation, increased follicular diameter as well as fully grown oocyte (>110 μm) viability and meiosis resumption. The oocytes from the cultured follicles were in vitro-matured and processed for fluorescence or ultrastructural analysis. The results showed that on Day 18 the treatment FF0-18 had a significantly higher (P<0.05) survival than control and FF12-18, but not FF6-18. The addition of bFF at the beginning of culture (FF0-18 and FF6-18) promoted a high percentage of follicular growth, meiosis resumption and early antrum formation. Moreover, this study described for the first time the ultrastructural analysis of caprine oocytes grown in vitro. This evaluation revealed that in the presence of bFF on (FF0-18) the in vitro-grown oocytes presented normal organelle distribution and well-defined, intact plasma and nuclear membranes. In conclusion, bFF originating from dominant follicles maintain the survival and promote the in vitro growth of goat preantral follicles when added at the beginning of culture.

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with ≥ 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39°C, whereas in the HS group, embryos were subjected to 41°C for 12 h, and then returned to 39°C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N × N; H × H; and H × GIR) and in both time intervals (4.0 and 6.5 h). However, N × N had higher cleavage rate (P < 0.05) in both time intervals when compared with H × H and H × GIR. In addition, Nelore oocytes fertilized with Nelore semen (N × N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H × H and H × GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to evaluate the effects of plasma progesterone (P4) concentrations and exogenous eCG on ovulation and pregnancy rates of pubertal Nellore heifers in fixed-time artificial insemination (FTAI) protocols. In Experiment 1 (Exp. 1), on Day 0 (7 d after ovulation), heifers (n = 15) were given 2 mg of estradiol benzoate (EB) im and randomly allocated to receive: an intravaginal progesterone-releasing device containing 0.558 g of P4 (group 0.5G, n = 4); an intravaginal device containing 1 g of P4 (group 1G, n = 4); 0.558 g of P4 and PGF(2α) (PGF; 150 μg d-cloprostenol, group 0.5G/PGF, n = 4); or 1 g of P4 and PGF (group 1G/PGF, n = 3). On Day 8, PGF was given to all heifers and intravaginal devices removed; 24 h later (Day 9), all heifers were given 1 mg EB im. In Exp. 2, pubertal Nellore heifers (n = 292) were treated as in Exp. 1, with FTAI on Day 10 (30 to 36 h after EB). In Exp. 3, pubertal heifers (n = 459) received the treatments described for groups 0.5G/PGF and 1G/PGF and were also given 300 IU of eCG im (groups 0.5G/PGF/eCG and 1G/PGF/eCG) at device removal (Day 8). In Exp. 1, plasma P4 concentrations were significantly higher in heifers that received 1.0 vs 0.588 g P4, and were significantly lower in heifers that received PGF on Day 0. In Exp. 2 and 3, there were no significant differences among groups in rates of ovulation (65-77%) or pregnancy (Exp. 2: 26-33%; Exp. 3: 39-43%). In Exp. 3, diameter of the dominant ovarian follicle on Day 9 was larger in heifers given 0.558 g vs 1.0 g P4 (10.3 ± 0.2 vs 9.3 ± 0.2 mm; P < 0.01). In conclusion, lesser amounts of P4 in the intravaginal device or PGF on Day 0 decreased plasma P4 from Days 1 to 8 and increased diameter of the dominant follicle on Day 9. However, neither of these nor 300 IU of eCG on Day 8 significantly increased rates of ovulation or pregnancy., (Copyright © 2011. Published by Elsevier Inc.)

The study evaluated, in early post-partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre-ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF(2)α and prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 IU of eCG, immediately after PGF(2)α treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 ± 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 ± 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14-dihydro-15-keto prostaglandin F(2)α (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre-ovulatory period was not effective in inhibiting PGFM release, which was lower in P4-primed than in non-primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4-primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming., (© 2009 Blackwell Verlag GmbH.)

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.

The objective was to evaluate the effects of temporary calf removal (TCR), eCG administration, or both, in a progesterone-based protocol. Suckled Nellore cows (40-80 d postpartum, n=443) with body condition scores from 2.0 to 3.5 (5-point scale) on three farms were all given a synchronizing protocol (PEPE). At the start (designated Day 0), cows were given an intravaginal device (1.0 g of progesterone) and 2.5mg of estradiol benzoate (EB) im. On Day 8, the device was removed and cows were given PGF(2 alpha) (150 microg of D-cloprostenol im), followed in 24h by 1.0mg EB im, and 30-36 h thereafter, fixed-time AI. The design was a 2 x 2 factorial; main effects were TCR (54-60 h; from device removal to FTAI) and eCG treatment (300 IU im, concurrent with PGF(2 alpha)). Transrectal ultrasonography was done on Days -10 and 0 to detect anestrus (absence of a CL at both examinations) and approximately 30 d after FTAI (pregnancy diagnosis). Data were analyzed by logistic regression. The following variables did not significantly affect pregnancy rates: farm, postpartum interval, cyclicity, inseminators, and semen (sire). Overall, 77% of the cows were deemed anestrus. Pregnancy rates were similar (P>0.05) among treatment groups: Control (54/108=50.0%), TCR (44/106=41.5%), eCG (63/116=54.3%), and TCR+eCG (49/113=43.4%). Pregnancy rate was higher in multiparous than primiparous cows (186/360, 51.7% vs. 24/83, 28.9%, P<0.01), but was not significantly affected by cyclicity status or body condition score. In conclusion, temporary calf removal, eCG, or both, did not significantly increase pregnancy rate to timed-insemination in a progesterone-based synchronization protocol in postpartum Nellore cows with acceptable body condition.