124 IDENTIFICATION OF LUTEINIZING HORMONE RECEPTOR ISOFORMS DURING FOLLICLE DEVELOPMENT IN CATTLE

2014 ◽  
Vol 26 (1) ◽  
pp. 176
Author(s):  
S. Wohlres-Viana ◽  
E. K. N. Arashiro ◽  
L. S. A. Camargo ◽  
C. A. C. Fernandes ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Full Length ◽  
Luteinizing Hormone Receptor ◽  
Frequency Of Occurrence ◽  
Follicle Diameter ◽  
Chi Squared ◽  
Exon 11 ◽  
Exon 10

The expression of the LH receptor (LHR) is required for the transition from FSH to LH-dependence during the establishment of follicular dominance in cattle. The aim of this study was to identify LHR isoforms expressed before, during, and after follicle deviation, using as models dairy breeds with different dominant follicle sizes at deviation. Mural granulosa cells (GC) were collected using an adapted ultrasound-guided follicular aspiration system (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175) from follicles of 6, 8, 10, or 12 mm in diameter of Holstein (Bos taurus), and of 4, 6, 8, or 10 mm of Gir (Bos indicus) heifers. The recovered follicular fluid was centrifuged and the cells were washed with NaCl 0.9% saline and kept in RNA Later (Ambion). Total RNA extraction was performed from GC using a commercial RNeasy Micro Kit (Qiagen), quantified in a spectrophotometer (Nanodrop), and reverse transcribed using the commercial Superscript III kit (Invitrogen). The generated cDNA were PCR amplified using a specific primer for the LHR and designed to detect a region of known occurrence of isoforms. The samples were previously tested for theca cell contamination using a primer to detect the CYP17A1 gene, and those showing contamination were excluded. Results of PCR were analysed by electrophoresis in 5% native acrylamide gel. The frequency of occurrence of the different isoforms was compared by the chi-squared test. In Holstein, the full-length form of the LHR mRNA (459 bp) was detected in all samples. The isoform with total deletion of exon 10 and partial of exon 11 (isoform II; 113 bp) was observed in 4 of 6 follicles of 6 mm and in 4 of 5 follicles of 8 mm. The isoform with total deletion of exon 10 (isoform III; 378 bp) was observed in 4 of 6 follicles of 6 mm, and in all follicles of 8 mm (5/5). The isoform with partial deletion of exon 11 (isoform IV; 194 bp) was observed in 4 of 6 follicles of 6 mm and in 4 of 5 follicles of 8 mm. These 3 alternative isoforms were present in all follicles of 10 mm (4/4) and 12 mm (11/11). There was no difference (P > 0.05) in the frequency of occurrence of the different isoforms. In Gir, the expression of LHR was less regular, no isoform was present in all samples, and no follicle size class showed all isoforms. The full-length LHR mRNA was detected in 2 of 7 follicles of 4 mm, 6 of 9 follicles of 6 mm, 2 of 6 follicles of 8 mm and in all follicles of 10 mm (6/6). The isoform II was observed in 3 of 7 follicles of 4 mm, 3 of 9 follicles of 6 mm, 2 of 6 follicles of 8 mm, and in all (6/6) follicles of 10 mm. The isoform III was observed in 6 of 7 follicles of 4 mm, 7 of 9 follicles of 6 mm, and in all follicles of 8 mm (6/6) and 10 mm (6/6). The isoform IV was observed in 5 of 7 follicles of 4 mm, 6 of 9 follicles of 6 mm, 5 of 6 follicles of 8 mm, and in 5 of 6 follicles of 10 mm. In the Gir breed, the isoform with deletion of exon 10 was the most frequent one (P < 0.01). More than one isoform was observed in most samples. In conclusion, 1) LHR is expressed in GC before follicle deviation, and 2) the expression of LHR isoforms is affected by follicle diameter and breed. The authors acknowledge support from CNPq 477701 and Fapemig PPM 0067/11.

251 FOLLICLE DEVIATION AND OVULATORY CAPACITY IN BOS INDICUS HEIFERS

2007 ◽  
Vol 19 (1) ◽  
pp. 242 ◽  
Author(s):  
L. U. Gimenes ◽  
N. A. T. Carvalho ◽  
M. F. Sá Filho ◽  
H. Ayres ◽  
J. R. S. Torres-Júnior ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Animal Health ◽  
Bos Indicus ◽  
Estradiol Benzoate ◽  
Transrectal Ultrasonography ◽  
Holstein Cows ◽  
Second Phase ◽  
Chi Square ◽  
Follicle Diameter ◽  
Two Phases

In Holstein cows, the diameter of the dominant follicle (DF) at the time of follicle deviation is 8.5 mm and the subordinate follicle (SF) is 7.2 mm (Ginther et al. 1996 Biol. Reprod. 55, 1187–1194). However, follicular responsiveness to an ovulatory treatment occurs only with 10.0-mm-diameter follicles (Sartori et al. 2001 Biol. Reprod. 65, 1403–1409). The current study tested the hypothesis that, in Bos indicus (Nelore and crossbred Nelore � Gir) females, the follicular diameters at the time of deviation and ovulation responsiveness are smaller than those in Holstein cows. The experiment was performed in two phases. In the first phase, 12 Nelore heifers were previously synchronized with a protocol using progestagen and estradiol benzoate. After implant removal, all heifers were evaluated by transrectal ultrasonography (Aloka SSD-500, Tokyo, Japan) every 12 h until Day 5 of the estrous cycle (Day 0 = Day of the ovulation) to assess the time of ovulation, the time of follicle deviation, and the follicular diameter at the deviation. In the second phase, 29 Bos indicus heifers (Nelore and crossbred Nelore � Gir) were previously synchronized with the same protocol as cited above. After the ovulations (Day 0), the follicles were evaluated by transrectal ultrasonography every 24 h, until they reached the diameter of 7.0–8.4 mm (n = 9); 8.5–10.0 mm (n = 10); and &gt;10.0 mm (n = 10). In order to assess the ovulatory capacity, all animals were treated with 25 mg of LH (Lutropin-V�; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) at these follicle diameter ranges. After the LH treatment, all animals were monitored by ultrasonography every 12 h for 48 h. ANOVA, Bartlett, and chi-square tests were used in the statistical analyses. In the first phase, the diameters of the DF and SF at the time of follicular deviation (61.9 � 4.9 h after ovulation) were 6.2 � 0.2 and 5.8 � 0.2 mm, respectively. In the second phase, the the average follicular diameters at the time of LH administration in the groups 7.0–8.4 mm, 8.5–10.0 mm, and &gt;10.0 mm were 7.6a � 0.1 mm, 9.6b � 0.1 mm, and 10.9c � 0.2 mm; and their ovulation rates were 33.3%a (3/9), 80.0%b (8/10), and 90.0%b (9/10), respectively (P &lt; 0.05). The interval from LH treatment to ovulation was 38.0 � 4.0 h, 31.5 � 2.7 h, and 30.0 � 2.0 h, respectively (P &gt; 0.05). In conclusion, in Bos indicus heifers, follicle deviation occurred with smaller diameters than previously reported in Bos taurus breeds. In addition, Bos indicus heifers are able to ovulate in response to 25 mg of LH with smaller diameters compared to those of Bos taurus breeds. Moreover, in Bos indicus heifers, ovulatory capacity is acquired by follicles as small as 7.0–8.4 mm, but this responsiveness significantly increases after follicles reach 8.5–10.0 mm. This work was supported by FAPESP (Proc:03/10203-4); Bioniche Animal Health, Inc., Belleville, Ontario, Canada; and Tecnopec, S�o Paulo, Brazil.

347 COULD THE DIFFERENTIAL EXPRESSION OF LUTEINIZING HORMONE RECEPTOR ISOFORMS EXPLAIN THE VARIABILITY IN SUPEROVULATORY RESPONSES IN CATTLE?

2015 ◽  
Vol 27 (1) ◽  
pp. 261
Author(s):  
S. Wohlres-Viana ◽  
E. K. N. Arashiro ◽  
J. G. V. Grazia ◽  
L. S. A. Camargo ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Bos Indicus ◽  
Luteinizing Hormone Receptor ◽  
Poor Responders ◽  
Embryo Production ◽  
Lh Receptor ◽  
Follicular Wave ◽  
Follicular Aspiration ◽  
Receptor Isoforms

Embryo production in vivo is highly variable among donors. The Gir breed (Bos indicus) is well known to show a low embryo production after superovulation (2.5 to 3.5 viable embryos per flush), and a high variance in superovulatory responses, which makes this breed an interesting model to study this trait. The aim of this study was to evaluate the expression pattern of LHR isoforms in Gir heifers previously characterised as good (10.3 ± 1.2 embryos/flush, N = 5) or poor (1.1 ± 0.3 embryos/flush, N = 5) responders to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175) was used to collect granulosa cells (GC) from 8-mm follicles growing in either a synchronized but not stimulated follicular wave (FW) or in the fourth day of superovulation (SOV), induced with 200 UI of FSHp (Pluset, Serono). The recovered follicular fluid was centrifuged and the cells were washed with NaCl 0.9% saline and kept in RNA Later (Ambion, Austin, TX, USA). Total RNA extraction was performed using the commercial RNeasy Micro Kit (Qiagen, Valencia, CA, USA). The RNA samples were quantified and reverse transcribed using the commercial Superscript III kit (Invitrogen, Carlsbad, CA, USA). Complementary DNA samples were amplified through real-time PCR, using a LH receptor primer – not selective for LHR isoforms (total LHR) – and 4 sets of isoform selective primers (S1, S10, S10+11, and S11). All samples were previously tested for theca cell contamination through detection of CYP17A1 gene, and those showing contamination were excluded. The β-actin gene was used as endogenous control. Analyses were performed using the REST software and the expression values are shown as mean ± s.e.m. For comparisons between good and poor responders, the first was set as 1.00. For comparisons between FW and SOV, FW was set as 1.00. In the good responder group, there was no difference (P > 0.05) in total LHR expression among GC samples from FW and SOV. However, the S10+11 isoform was down-regulated (0.4 ± 0.1; P < 0.01) after SOV. In the poor responders group, total LHR expression was down-regulated (0.2 ± 0.1; P < 0.01) after SOV, but there was no difference in the expression of isoforms (P > 0.05). Contrasting the response groups (good and poor), total LHR (15.1 ± 7.6; P < 0.001), and the isoforms S10 (5.7 ± 2.7; P < 0.01), S10+11 (1.9 ± 0.6; P < 0.01), and S11 (5.1 ± 2.5; P < 0.01) were up-regulated in FW of poor responders, but there was no difference (P > 0.05) in any LHR form during SOV. We concluded that 1) LHR expression is different between heifers characterised as good or poor responders to superovulation; 2) superovulation modulates the LHR expression and reduces the original differences observed in unstimulated cycles; 3) diminished expression of total LHR, but not in the isoforms, in poor responders heifers could suggest a reduction in the expression of full-length LHR, with possible consequences to ovulatory capability after superovulation.Financial support was provided by CNPq Project 477701 and Fapemig PPM 0067/11.

scholarly journals Follicular dynamic and repeatability of follicular wave development in Peranakan Ongole (PO) cattle

2016 ◽  
Vol 21 (1) ◽  
pp. 26 ◽  
Author(s):  
Muhammad Imron ◽  
Iman Supriatna ◽  
. Amrozi ◽  
Mohamad Agus Setiadi
Keyword(s):  
Bos Taurus ◽  
Research Result ◽  
Bos Indicus ◽  
Follicular Development ◽  
Wave Pattern ◽  
Follicular Wave ◽  
Follicle Diameter ◽  
Wave Patterns ◽  
The Difference ◽  
Follicular Dynamics

<p class="abstrak2">Superovulation treatment on PO cattle (Bos indicus) was less responsive compared to Bos taurus breed. It might due to the difference of their follicular dynamic. This study was conducted to investigate the follicular dynamics and its repeatability in PO cattle. Follicular dynamics observations conducted on 9 cows trough ultrasound scanning every day. Observations of wave patterns repeatability were performed in 6 cows which its wave pattern already known on the next consecutive IOI.  Research result indicated that PO cattle had 3 (66%) and 4-waves (34%) pattern. The first wave of 3 and 4-waves pattern emerged on day -0.4+0.9 and 1.4+1.1 respectively.  The second wave of 3 and 4-wave pattern emerged on day 9.8+1.5 and 7.4+1.9 respectively.  The pattern of 3 waves has a longer follicle dominant duration (11.6+1.5 day) in the first wave of estrous cycle, compared with 4 waves pattern (10+2.92 and 7+1.00 day respectively). The growth rate of dominant follicle was not different significantly between the 3 and 4-waves pattern (0.87+0.23 and 0.94+0.25 mm/day respectively). Similarly, ovulatory follicle diameter between 3 and 4-waves pattern was also not different significantly (12.24+12.34 and 12.30+12.23 mm respectively). Observation of wave patterns repeatability in 6 PO cows indicated that PO cattle had high repeatability in follicular wave pattern (0.88) and the number of growing follicle was 0.91.  This study resulted data for dynamic of follicular development, wave pattern, its repeatability which be expected to design the protocol of superovulation treatment or other reproduction technologies based on follicular dynamic to improve its result in PO cattle.</p><p> </p>

230 EXPRESSION OF mRNA ENCODING FGF10 AND COGNATE RECEPTORS (FGFR1B AND FGFR2B) AROUND FOLLICLE DEVIATION IN NELORE HEIFERS

2010 ◽  
Vol 22 (1) ◽  
pp. 273
Author(s):  
A. C. S. Castilho ◽  
M. F. Machado ◽  
D. M. Guerra ◽  
R. Ereno ◽  
C. M. Barros ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Mrna Abundance ◽  
Morphological Divergence ◽  
Dominant Follicle ◽  
Ultrasound Monitoring ◽  
Follicle Diameter ◽  
Follicle Selection

A member of the FGF7 subfamily, FGF10 acts via FGFR2B and FGFR1B. In bovine antral follicles, FGF-10 was detected in oocytes and theca cells (TC). Levels of mRNA were negatively correlated with intrafollicular concentrations of estradiol, and FGF10 inhibited estradiol production from granulosa cells (GC). In Nellore (Bos indicus), morphological divergence occurs on average 2.5 days after ovulation, when dominant follicle diameter is around 6.0 mm. To gain insight into the involvement of the FGF10 system in the control of follicle selection, we assessed mRNA expression of FGF10 in TC and of FGFR1B and FGFR2B in GC from dominant and subordinate follicles around deviation in Nellore heifers. Thirteen Nellore heifers were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (n =4), 2.5 (n = 5), and 3 (n = 4) days after ovulation. Granulosa cells and TC were separated from the 2 largest follicles and submitted to total RNA extraction. mRNA abundance of CYP19 (aromatase), FGF10, FGFR1B, and FGFR2B was measured by real-time RT-PCR and normalized by the expression of cyclophilin A (CYCA) and GAPDH, for TC and GC, respectively. Dominant and subordinate follicles were considered those expressing the greatest and second-greatest abundance of CYP19 mRNA in GC within each heifer. Effects of follicle status and day on CYP19, FGF10, FGFR2B, and FGFR1B mRNA abundance were tested by ANOVA. On Day 2, FGFR2B mRNA abundance was greater in GC of subordinate follicles compared with dominant follicles (P = 0.006), and that of FGF10 in TC tended to exhibit the same pattern (P = 0.06). Follicle diameter was not different between dominant and subordinate follicles on Day 2 (5.5 ± 0 v. 5.12 ± 0.3 cm). On Day 2.5, FGF10 expression was greater in TC from subordinate follicles (P = 0.01), and FGFR2B expression in GC was no longer different between dominant and subordinate follicles. Follicle diameter was greater in dominant follicles on Day 2.5 (6.7 ± 0.2 v. 5.8 ± 0.3 cm; P = 0.04). On Day 3, no differences were observed between dominant and subordinate follicles for any of the genes assessed. mRNA expression of FGFR1B in GC did not change with follicle status or day. In conclusion, expression of FGF10 and FGFR2B was decreased in dominant follicles around morphological divergence, suggesting their involvement in the mechanisms controlling dominant follicle selection. As FGF10 inhibits estradiol production of GC, we propose that FGF10 and FGFR2B are suppressed in the dominant follicle to allow acquisition of full steroidogenic capacity. This research was supported by FAPESP.

178 EXPRESSION OF mRNA ENCODING LUTEINIZING HORMONE RECEPTOR AND MEVALONATE KINASE AROUND FOLLICLE DEVIATION IN NELORE HEIFERS (BOS INDICUS)

2013 ◽  
Vol 25 (1) ◽  
pp. 238
Author(s):  
A. C. Souza Castilho ◽  
R. L. Ereno ◽  
M. Fernandes Machado ◽  
R. A. Satrapa ◽  
M. F. Gouveia Nogueira ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Luteinizing Hormone Receptor ◽  
Mrna Abundance ◽  
Mevalonate Kinase ◽  
Dominant Follicle ◽  
Significant Difference

Luteinizing hormone (LH) plays a key role in controlling physiological processes in the ovary, and the expression of LHR by bovine granulosa cells is crucial to the follicular transition from FSH to LH dependency. There are controversies about the time at which follicles acquire LHR in granulosa cells. In Nelore breed (Bos indicus), the morphological divergence occurs, on average, 2.5 days after ovulation when the diameter of the dominant follicle is ~6.0 mm. In our previous work with semiquantitative PCR, the mRNA expression of LHR isoforms was detected more clearly after deviation (Day 3). The LHR mRNA binding protein, mevalonate kinase (MVK), is responsible for the down-regulation of LHR mRNA, thereby controlling the steady-state of LHR mRNA expression. In rats, there is an inverse correlation between the mRNA expression of LHR and MVK in luteal cells; however, there is no evidence about MVK expression in the bovine antral follicle. To gain insight about the involvement of the LHR/MVK system in the control of follicle deviation, we assessed the mRNA expression of LHR and MVK in granulosa cells from dominant and subordinate follicles close to deviation in Nelore heifers. Animals (n = 10) were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (before deviation; n = 3), 2.5 (around deviation; n = 4), and 3 (post-deviation; n = 3) days after ovulation. Granulosa cells were harvested from the 2 largest follicles and submitted to total RNA extraction and reverse transcribed with oligo-dT. The mRNA abundance of LHR and MVK was measured by real-time RT-PCR using the Sybr Green system with bovine-specific primers and normalized by the expression of endogenous gene, cyclophilin A (PPIA), using the ΔΔct method corrected by Pffafl’s equation. Dominant and subordinate follicles were considered those expressing the greatest and second greatest abundance of aromatase mRNA (CYP19) in granulosa cells within each heifer. Effects of the day and follicle status on the mRNA abundance of LHR and MVK were tested by ANOVA and the mean values compared by paired t-test or Tukey test (P < 0.05 indicated significant difference). The LHR mRNA was detected at the predicted time of follicle deviation in Nelore heifers (Day 2.5) and was higher in dominant follicle on Day 3 (32.8 ± 12.6) compared with Day 2.5 (3.2 ± 0.9). The second largest follicle (subordinate follicles) had lower mRNA abundance of LHR when compared with future dominant follicles (largest follicles) on days 2.5 (0.8 ± 0.4 v. 3.2 ± 0.9) and 3 (1.9 ± 0.8 v. 32.8 ± 12.6). In contrast to the mRNA expression of LHR, MVK mRNA was more expressed in the subordinate follicles than in the largest follicles at Days 2.5 (3.1 ± 0.9 v. 0.9 ± 0.3) and 3 (2.6 ± 0.6 v. 0.9 ± 0.1) after ovulation, suggesting that it may be necessary to decrease the MVK expression in future dominant follicles to increase their LHR expression and follow up to ovulation. Supported by FAPESP.

172 P450 AROMATASE GENE EXPRESSION, ESTRADIOL PRODUCTION, AND DOMINANT FOLLICLE DEVIATION IN BOS TAURUS AND BOS INDICUS DAIRY HEIFERS

2013 ◽  
Vol 25 (1) ◽  
pp. 234
Author(s):  
E. K. N. Arashiro ◽  
S. Wohlres-Viana ◽  
M. P. Palhao ◽  
L. S. A. Camargo ◽  
M. Henry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Dominant Follicle ◽  
Aromatase Gene ◽  
P450 Aromatase ◽  
Dairy Heifers ◽  
Follicular Waves ◽  
Aromatase Gene Expression

It is well documented that the size of the dominant follicle at deviation is smaller in Bos indicus compared with in Bos taurus breeds. The physiological mechanisms underlying this difference, however, are unknown. The aim of the present study was to evaluate the dynamic of oestradiol production during follicle development close to the expected moment of deviation in Bos taurus and Bos indicus dairy heifers. Intrafollicular concentration of oestradiol (E2) and P450 aromatase gene expression in granulosa cells (GC) were evaluated in Gir (n = 10) and Holstein (n = 10) heifers. Follicular waves were synchronized with an intravaginal progesterone device (1 g, Sincrogest, Ourofino Agropecuária, São Paulo, Brazil) and benzoate oestradiol (2 mg im, Sincrodiol, Ourofino Agropecuária). Ultrasonography evaluations (MyLab30 Vet Gold, Esaote, Genova, Italy, with a 7.5-MHz transducer) were performed every 24 h to detect the emergence of the new follicular waves. The largest follicle of each wave was individually aspirated by ovum pickup before, at the expected diameter, or after deviation in both Gir (4.6 ± 0.2, 6.3 ± 0.2, and 8.5 ± 0.6 mm, respectively) and Holstein heifers (6.0 ± 0.5, 8.6 ± 0.4, and 10.2 ± 0.2 mm, respectively), as previously described (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175). Follicular fluid (FF) samples were centrifuged and the supernatant stored at –20°C until E2 and progesterone (P4) determination by RIA. The pellet of GCs was washed twice with PBS, kept in RNAlater, and frozen at –20°C until RNA extraction and reverse transcription. Relative transcript quantification was performed by real-time PCR. The β-actin gene was used as control. Samples of FF with E2:P4 ratio <1 or presenting contamination by theca cells (detected by the expression of 17α-hydroxylase) were not used for statistical analyses. Concentration of E2 in FF was evaluated between breeds and among follicle size classes by ANOVA and differences among means compared by Student t-test or Tukey’s test, respectively. Within breeds, relative gene expression was accessed by pair-wise fixed reallocation randomization test (software REST®). Results are shown as mean ± SEM. In both breeds, concentration of E2 in FF progressively increased with follicular diameter (P < 0.05). Intrafollicular concentration of E2 (ng mL–1) was greater (P < 0.05) in Holstein than in Gir before (58.5 ± 11.7 v. 8.8 ± 2.0), at expected (226.0 ± 49.9 v. 78.9 ± 21.0), and after follicle deviation (579.1 ± 45.0 v. 185.0 ± 34.9). Interestingly, however, follicles with similar diameters (~6 or 8 mm) showed similar (P > 0.05) E2 concentrations between Holstein and Gir. Moreover, in both breeds, the relative expression of P450 aromatase gene in GC first increased (3.9 ± 2.4 and 67.5 ± 52.8 for Holstein and Gir, respectively; P < 0.05) at the same stage of follicular development (8 mm). The present results suggest that the smaller size of follicles at deviation in Bos indicus is not related to an earlier increase in intrafollicular E2 production. CNPq, CAPES, and Fapemig (CVZ APQ 02863/09).

262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE

2007 ◽  
Vol 19 (1) ◽  
pp. 247
Author(s):  
L. S. A. Camargo ◽  
J. H. M Viana ◽  
R.V. Serapião ◽  
M. F. M. Guimarães ◽  
W. F. Sá ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Real Time ◽  
Real Time Pcr ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Tropical Climate ◽  
Relative Quantification ◽  
Vitro Fertilization

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P &lt; 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes. Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.

199 EFFECT OF PRODUCTION SYSTEM AND BREED ON RELATIVE GENE EXPRESSION IN BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 198 ◽  
Author(s):  
S. Wohlres-Viana ◽  
M. C. Boite ◽  
M. M. Pereira ◽  
W. F. Sa ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Production System ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
In Vitro Production ◽  
System Effect ◽  
Vitro Production

Embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. Nevertheless, there are a few studies showing the effect of in vitro environment on embryos from different bovine subspecies, such as Gyr (Bos indicus) and Holstein (Bos taurus). The aim of this study was to evaluate the relative abundance of aquaporin 3 (AQP3) and ATPase-α1 (Na/K-ATPase alpha 1) transcripts in blastocysts produced in vivo or in vitro from Gyr and Holstein cattle. The production system effect (in vivo × in vitro) for Gyr cattle and the breed effect (Holstein × Gyr) for in vitro-produced embryos were evaluated. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA). The cDNA obtained were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed with REST software© using the pair wise fixed reallocation randomization Test. There was no difference (P > 0.05) in gene expression for AQP3 and ATPase-α1 between in vivo- and in vitro-produced Gyr embryos, although the results suggest that the APQ3 gene was down-regulated (0.81 ± 0.31) and the ATPase-α1 gene was up-regulated (1.20 ± 0.65) in embryos produced in vitro. For breed effect within in vitro production system, ATPase-α1 gene was down-regulated in Holstein (0.56 ± 0.30) when compared with Gyr embryos (P < 0.05). The same trend was observed for AQP3 (0.58 ± 0.25), but with no difference (P > 0.05). In conclusion, the data suggest that embryo production system does not interfere with the transcript amount of the genes studied for Gyr cattle; however, the in vitro production system may have different effects on gene expression according to embryo breed. Other genes should be evaluated for a better understanding of these differences. Financial support: CNPq, Fapemig.

239 INFLUENCE OF BREED PREDOMINANCE ON MATURATION COMPETENCE, FERTILIZATION, AND DEVELOPMENT IN VITRO OF BOVINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
J. Pelaez ◽  
H. Hernandez-Fonseca ◽  
A. Pirela ◽  
F. Baez ◽  
P. Villamediana ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Cleavage Rate ◽  
Ethanol Acetic Acid ◽  
Chi Squared ◽  
Development In Vitro ◽  
Bovine Oocytes

The purpose of this research was to compare the competence of bovine oocytes of different breed predominance (Bos taurus v. Bos indicus) to mature and to be fertilized. This was done through the collection, selection, maturation, and fertilization of oocytes from slaughtered cows, predominantly either B. taurus or B. indicus. Only cows that were at least 5/8 B. taurus or 5/8 B. indicus, according to a series of phenotypic characteristics, such as the presence of a hump, dewlap, length of the ears, and others, were selected. To obtain cumulus–oocyte complexes, ovarian follicles (3 to 10 mm in diameter) were aspirated, and only oocytes with 2 or more layers of cumulus cells, an intact zona pellucida, and a homogeneous granular cytoplasm were selected. After selection, oocyte maturation [in vitro maturation (IVM)] and IVF were done. Frozen–thawed semen was used from one Brahman bull (B. indicus). For the evaluation of IVM as for IVF, oocytes were fixed for approximately 24 h at 4°C in a solution of ethanol : acetic acid (3 : 1). They were then stained with 1% acetic orcein. A chi-squared test was performed for all reported rates. The rate of maturation of oocytes from cows with a predominant B. indicus phenotype was 66.93%, whereas cows with a B. taurus phenotype reached 43.10% (P < 0.001). Regarding the fertilization rate, predominantly B. indicus females had 43.68% of oocytes normally fertilized and 41.74% of oocytes were abnormally penetrated. This category included polyspermic and asynchronic (abnormally developed pronucleus) oocytes. In cows with B. taurus predominance, 31.96% of oocytes were normally penetrated and 46.39% were abnormally penetrated by spermatozoa (no significant differences were found). The rate of non-fertilized oocytes was significantly different (P < 0.05) among B. indicus and B. taurus oocytes (6.79 and 17.52%, respectively). A small and nonsignificant proportion of degenerated oocytes resulted in both groups (7.79% for B. indicus and 4.14% for B. taurus). The cleavage rate was not different among phenotypic groups (36.12 and 32.30%, respectively, for B. indicus and B. taurus). In conclusion, the present results indicate that oocytes from predominantly B. indicus cows were more competent than oocytes from cows with a predominance of the B. taurus breed. Nonetheless, this superiority was not evident in terms of cleavage rates. Semen from other B. indicus and B. taurus breeds must be tested to clarify any breed interactions.

318 EFFECT OF MATERNAL HEAT STRESS ON OOCYTE QUALITY AND IN VITRO COMPETENCE IN BOS INDICUS CATTLE

2007 ◽  
Vol 19 (1) ◽  
pp. 274
Author(s):  
J. R. S. Torres Júnior ◽  
M. F. A. Pires ◽  
W. F. Sá ◽  
A. M. Ferreira ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bos Indicus ◽  
Oocyte Quality ◽  
Blastocyst Development ◽  
Estrous Cycles ◽  
Follicle Diameter ◽  
Chi Squared ◽  
Sixth Session ◽  
Neutral Conditions

High temperatures can be harmful to the competence of cumulus–oocyte complexes and to embryo development (Al-Katanani et al. 2002 J. Dairy Sci. 85, 390–396). The aim of this study was to evaluate the effect of maternal heat stress on in vitro embryo yield. Ten multiparous nonlactating Gir (Bos indicus) cows were kept in tie stalls for an adaptive period of 28 days [pre-heat-stress period (PRE-HS)/Days -28 to -1]. Cows were subjected to 2 OPU (ovum pickup) sessions (Days -14 and -7). In the heat-stress period (HS; Days 0 to 28), cows were divided into control (C: n = 5) and heat-stressed (HS: n = 5) groups. During this period, OPU sessions were performed once a week from Days 0 to 28. The C group remained in a thermo-neutral environment, and the HS group was kept in a climatic chamber with controlled temperature and humidity (38�C and 80% during the day and 30�C and 80% during the night). In the post-heat-stress period (POST-HS; Days 28 to 147), all cows returned to thermo-neutral conditions. Then 17 OPU sessions were performed once a week from Days 35 to 147. In all periods, blood samples were collected weekly for progesterone (P4) analysis, and ovarian follicles were counted, measured, and aspirated. The COCs were evaluated and selected for the IVF procedure. Data were analyzed by ANOVA (PROC MIXED of SAS) and a chi-squared test. The luteal phase was defined as the period between 2 samples with P4 below 1.0 ng mL-1. A handling accident caused the exclusion of an HS cow after the sixth session. The C and HS groups were subjected to 125 and 107 OPU sessions, respectively. Means � SEM for the C vs. HS groups, in the PRE-HS, HS, and POST-HS periods, respectively, were visualized follicles: 25.5 � 2.5 vs. 28.5 � 2.8, 24.2 � 1.1 vs. 24.0 � 1.9, and 15.3 � 0.6 vs. 15.8 � 0.8; largest follicle diameter: 12.1 � 1.5 vs. 11.1 � 1.7, 13.3 � 0.8 vs. 13.0 � 0.6, and 11.4 � 0.4b vs. 14.0 � 0.4a; P &lt; 0.05; 2nd largest follicle diameter: 6.2 � 1.3 vs. 6.0 � 1.2, 5.9 � 0.6 vs. 7.1 � 0.8, and 6.3 � 0.3b vs. 8.7 � 0.5a; recovered COCs: 11.2 � 2.8 vs. 14.3 � 2.5, 9.6 � 1.0 vs. 11.0 � 1.3, and 8.6 � 0.7 vs. 7.9 � 0.6; COCs selected for IVF: 69/112 (61.6%)b vs. 108/143 (75.5%)a, 164/241 (68%) vs. 172/265 (64.9%), and 426/712 (59.8%) vs. 305/535 (75.0%); cleavage: 44/59 (74.5%) vs. 87/105 (82.9%), 72/101 (71.3%) vs. 74/121 (61.2%), and 226/317 (71.3%) vs. 159/230 (69.1%); embryos per cow/OPU: 2.1 � 1.1y vs. 4.1 � 1.0x, 0.4 � 0.3 vs. 0.5 � 0.3, and 0.9 � 0.2x vs. 0.4 � 0.1y; P &lt; 0.1; and blastocyst yield: 16/59 (27.1%) vs. 33/105 (31.5%), 11/31 (35.5%) vs. 13/52 (25.0%), and 76/279 (27.2%)a vs. 25/188 (13.3%)b. In conclusion, maternal heat stress increased the percentage of short estrous cycles, decreased the P4 concentrations, and decreased the number of embryos produced by Bos indicus cows, mainly from 28 to 147 days post-heat-stress, showing long-term deleterious effects on blastocyst development.

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