scholarly journals Effects of oestradiol and progesterone on secretion of gonadotrophins and health of first wave follicles during the oestrous cycle of beef heifers

Reproduction ◽  
2002 ◽  
pp. 531-541 ◽  
Author(s):  
EJ Austin ◽  
M Mihm ◽  
AC Evans ◽  
JL Ireland ◽  
JJ Ireland ◽  
...  
Keyword(s):  
Growth Factor ◽  
Antral Follicle ◽  
Follicle Development ◽  
Dominant Follicle ◽  
Beef Heifers ◽  
Follicle Diameter ◽  
Oestradiol Benzoate ◽  
Lh Release ◽  
Follicle Selection ◽  
Intravaginal Device

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.

109 Pre-synchronization and reutilization of progesterone devices during a 6-day CO-Synch protocol for fixed-time artificial insemination in beef heifers

2021 ◽  
Vol 33 (2) ◽  
pp. 161
Author(s):  
E. R. Canadas ◽  
B. J. Duran ◽  
G. Machado ◽  
A. Nall ◽  
S. E. Battista ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Mixed Models ◽  
Generalized Linear Mixed Models ◽  
Fixed Time ◽  
P Value ◽  
Beef Heifers ◽  
Follicle Diameter ◽  
Follicle Size ◽  
Gonadotrophin Releasing Hormone ◽  
Intravaginal Device

Ovulatory response to the initial gonadotrophin-releasing hormone (GnRH) of the CO-Synch protocol is affected by circulating progesterone (P4) and follicle size. In addition, heifers that ovulate to the initial GnRH treatment have greater fertility after AI. Thus, this study determined the effect of (1) presynchronization (Presynch) before a 6-day CO-Synch protocol and (2) circulating [RCE1] (P4) on ovulatory response, oestrus expression, and pregnancies per AI (P/AI) in beef heifers. Yearling beef heifers (n=233) at three locations were randomly assigned in a 2×2 factorial design to the following treatments: (1) Presynch+6-day CO-Synch with a new P4 device; (2) Presynch+6-day CO-Synch with a once-used P4 device; (3) 6-day CO-Synch with a new P4 device; and (4) 6-day CO-Synch with a once-used P4 device. Presynch consisted of insertion of a new P4 intravaginal device (1.38g of P4) on Day −17 and removal of P4 device on Day −11 concurrently with 500µg of cloprostenol sodium (PGF). On Day −9, all heifers received either a new (New) or once-used (Used) CIDR and 100µg of gonadorelin acetate (GnRH). Six days later (Day −3), CIDRs were removed, 1000µg of PGF was administered and an oestrous detection patch applied (Estrotect). At 72h after CIDR removal, AI was performed concurrently with administration of 100µg of GnRH. Pregnancy was determined by transrectal ultrasonography 31 days after AI. A subset of heifers (n=155) was examined on Day −9 and Day −3 by ultrasonography to determine ovulation to Day −9 GnRH. Data were analysed using generalized linear mixed models (SAS 9.4; SAS Institute Inc.). Presynch heifers had larger follicle diameter on Day −9 (12.7±0.3 vs. 10.1±0.3 mm; P<0.001), greater ovulatory response to Day −9 GnRH (82.5%; 66/80 vs. 56%; 42/75; P<0.001), greater expression of oestrus (90.6%; 106/117 vs. 78.4%; 91/116; P<0.02), and earlier oestrus (49.8±1 vs. 53.1±1 h; P<0.01) compared with controls. There was a treatment×CIDR interaction on oestrous expression, such that a lesser (P<0.05) percentage of control heifers with new CIDR expressed oestrus compared with all other groups (Table 1). Heifers with a used CIDR during the 6-day CO-Synch tended (P=0.08) to have greater P/AI (52.1%; 61/117) than those with a new CIDR (40.5%; 47/116). In conclusion, presynchronization before initiation of a 6-day CO-Synch increased follicle diameter, enhanced ovulatory response and oestrous expression, but did not affect fertility. The earlier onset of oestrus in presynchronized heifers suggests that the timing of AI may need to be modified. Table 1. Oestrous expression and pregnancy per AI (P/AI) in beef heifers with or without presynchronization and treated with a new or used CIDR during a 6-day CO-Synch Treatment CIDR Oestrus (%; n/n) Time of oestrus (h) P/AI (%; n/n) Control New 67.8a (40/59) 53.7±1.5a 33.9 (20/59) Used 89.5b (51/57) 52.7±1.6a 50.9 (29/57) Presynch New 94.7b (54/57) 50.9±1.4b 47.4 (27/57) Used 86.7b (52/60) 48.7±1.3b 53.3 (32/60) P-value Treatment 0.03 0.01 0.21 CIDR 0.62 0.19 0.08 Interaction 0.003 0.75 0.38 a,bValues with different superscripts differ (P<0.05).

129 Ovarian dynamics and gonadotropins during selection of the dominant follicle in postpartum lactating versus non-postpartum cycling mares

2020 ◽  
Vol 32 (2) ◽  
pp. 191
Author(s):  
M. Pastorello ◽  
M. O. Gastal ◽  
G. K. Piquini ◽  
D. B. Godoi ◽  
E. L. Gastal
Keyword(s):  
Growth Rate ◽  
Maximum Diameter ◽  
Sequential Data ◽  
Dominant Follicle ◽  
Follicle Diameter ◽  
The Mean ◽  
Low Levels ◽  
Follicle Selection ◽  
Ovarian Dynamics ◽  
Selection Of

The mare, compared to other livestock, has the shortest interval from partum to the first ovulation. In monovulatory species, the follicle deviation process in a wave is characterised by the continued growth of the dominant follicle (DF) and regression of the subordinate follicle. Although follicle diameter deviation, a key event of follicle selection, has been investigated during the oestrous and menstrual cycles, the occurrence of this phenomenon before the first postpartum ovulation seems to be unclear in all species. This study aimed to compare the follicular dynamics and gonadotropin profiles around the follicle diameter deviation day in postpartum lactating (PP Lactating; n=24) versus non-postpartum cycling (NPP Cycling; n=15) mares. On the day of parturition, every PP Lactating mare was paired with an NPP Cycling mare, and ovarian follicles (>4mm) were tracked daily by transrectal ultrasonography, and blood samples were collected. Data were analysed in the PP Lactating group according to the length of the partum-ovulation interval (POI; ≤22 and >22 days) and the postpartum interovulatory interval (PPIOI), and in the NPP Cycling group during two interovulatory intervals (1st and 2nd IOI). In addition, regardless of group, all four intervals were compared. We performed the FSH and LH assays using radioimmunoassay. Ovarian and hormonal parameters were analysed using ANOVA for sequential data. The day and diameter of the DF at the deviation (overall mean: 14.9±2.5 days; 21.7±0.4mm, respectively) were not different (P>0.05) between PP Lactating and NPP Cycling mares. However, when considering the length of POI, follicle deviation occurred 4.4±0.8 days earlier (P<0.001) in mares with POI ≤ 22 days than in mares with POI>22 days. No difference was found between PP Lactating and NPP Cycling mares within and between groups for the intervals from deviation to maximum diameter of the DF (10.4±0.4 days), for the intervals from deviation to ovulation (12.1±0.5 days), or for the growth rates of the DF from deviation to maximum diameter (2.6±0.1mm per day). The growth rate of the DF from deviation to ovulation (2.4±0.1mm per day) did not differ between PP Lactating and NNP Cycling mares; however, this growth rate was lower (P<0.03) in the POI, PPIOI, and 1st IOI compared with the 2nd IOI. The mean diameter of the DF around deviation (days −3 to 3; 22.5±0.3mm), and systemic FSH (days −4 to 4; 10.3±0.2ngmL−1) were not different between PP Lactating and NPP Cycling mares. Level of LH was lower (P<0.0001) around deviation (days −4 to 4) in the PP Lactating (0.7±0.0ngmL−1) versus the NPP Cycling mares (1.8±0.1ngmL−1). Results demonstrated that a partum effect occurs only on the day of deviation in mares during the foal heat (POI ≤ 22 days) and that low levels of LH during both intervals in PP Lactating mares were not detrimental to prevent ovulation.

Identification of novel genes associated with dominant follicle development in cattle

2007 ◽  
Vol 19 (8) ◽  
pp. 967 ◽  
Author(s):  
Anna E. Zielak ◽  
Niamh Forde ◽  
Stephan D. E. Park ◽  
Fiona Doohan ◽  
Paul M. Coussens ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Follicular Development ◽  
Intracellular Signalling ◽  
Follicle Development ◽  
The Novel ◽  
Dominant Follicle ◽  
Novel Genes ◽  
Follicular Wave ◽  
New Genes ◽  
Follicle Selection

Follicle development is regulated by the interaction of endocrine and intrafollicular factors, as well as by numerous intracellular pathways, which involves the transcription of new genes, although not all are known. The aim of the present study was to determine the expression of a set of unknown genes identified by bovine cDNA microarray analysis in theca and granulosa cells of dominant and subordinate follicles, collected at a single stage of the first follicular wave using quantitative real-time polymerase chain reaction. Differences were further examined at three stages of the follicular wave (emergence, selection and dominance) and bioinformatics tools were used to identify these originally unknown sequences. The suggested name function and proposed role for the novel genes identified are as follows: MRPL41 and VDAC2, involved in apoptosis (dominant follicle development); TBC1D1 stimulates cell differentiation (growth associated with dominant follicle selection and development); STX7, promotes phagocytosis of cells (subordinate follicle regression); and SPC22 and EHD3, intracellular signalling (subordinate follicle regression). In conclusion, we have identified six novel genes that have not been described previously in ovarian follicles that are dynamically regulated during dominant follicle development and presumably help mediate intracellular signalling, cell differentiation, apoptosis and phagocytosis, events critical to follicular development.

scholarly journals Dynamic Changes in the Intrafollicular Inhibin/Activin/Follistatin Axis during Human Follicular Development: Relationship to Circulating Hormone Concentrations*

2000 ◽  
Vol 85 (9) ◽  
pp. 3319-3330
Author(s):  
Alan L. Schneyer ◽  
Toshihiro Fujiwara ◽  
Janis Fox ◽  
Corrine K. Welt ◽  
Judith Adams ◽  
...  
Keyword(s):  
General Pattern ◽  
Follicular Development ◽  
Antral Follicle ◽  
Specific Pattern ◽  
Follicle Development ◽  
Dominant Follicle ◽  
Human Follicular Fluid ◽  
Inhibin A ◽  
Normal Women ◽  
Orderly Transition

Abstract Previous studies of normal human ovaries suggest that inhibins, activins, and follistatin (FS) are produced in a stage-specific pattern indicative of intraovarian, autocrine/paracrine roles in regulating follicle development. However, these studies relied largely on surgical specimens and thus include little information about the menstrual cycle stage or dominant follicle status at the time follicles or ovaries were obtained. The purpose of this study was to 1) determine the pattern of intrafollicular hormone biosynthesis across antral follicle development in normal women, 2) compare hormone concentrations in dominant and nondominant follicles from the same ovary, and 3) examine the relationship between dominant follicle hormone content and circulating hormone levels. Intrafollicular estradiol, progesterone, and inhibin A concentrations increased significantly with follicle size or maturity, whereas significant inverse relationships were observed for androstenedione and the androstenedione/estradiol (A:E) ratio. In contrast, neither inhibin B, activin A, nor free FS varied consistently with size or maturity. Estradiol, progesterone, and inhibin A levels and A:E ratio were significantly lower in nondominant follicles compared to the dominant follicle aspirated from the same ovary. Although intrafollicular and serum concentrations of each hormone followed the same general pattern as follicles develop, the human follicular fluid/serum gradients changed during the follicular phase and were different for estradiol and inhibin A, suggesting the presence of stage-specific differences in pharmacodynamics. These results are consistent with the hypothesis that the orderly transition from an activin-dominant to an inhibin A/FS-dominant microenvironment is critical for dominant follicle development.

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.

115 Presynchronization and reutilization of progesterone devices during a 6-day CO-Synch protocol for fixed-time artificial insemination in beef heifers

2021 ◽  
Vol 33 (2) ◽  
pp. 165
Author(s):  
E. R. Canadas ◽  
B. Duran ◽  
G. Machado ◽  
A. Nall ◽  
S. E. Battista ◽  
...  
Keyword(s):  
Factorial Design ◽  
Artificial Insemination ◽  
Fixed Time ◽  
P Value ◽  
Beef Heifers ◽  
Follicle Diameter ◽  
Follicle Size ◽  
Gonadotrophin Releasing Hormone ◽  
Intravaginal Device ◽  
Second Use

Ovulatory response to the initial gonadotrophin-releasing hormone (GnRH) of the CO-Synch protocol is affected by circulating progesterone (P4) and follicle size. In addition, heifers that ovulate to initial GnRH treatment have greater fertility after AI. Thus, the aim of this study was to assess the effect of (1) presynchronization (Presynch) before a 6-day CO-Synch protocol and (2) P4 device reuse (new vs. second use) on ovulatory response, oestrous expression, and pregnancy per AI (P/AI) in beef heifers. Yearling beef heifers (n=233) were randomly assigned in a 2×2 factorial design to the following treatments: (1) Treatment (Presynch): (1a) Presynch+6-day CO-Synch with new P4 device; (1b) Presynch+6-days CO-Synch with a once-used P4 device; (2) Control (no Presynch): (2a) 6-day CO-Synch with new P4 device; (2b) 6-day CO-synch with once-used P4 device. Presynch consisted of insertion of a new P4 intravaginal device (CIDR, 1.38g of P4; Zoetis) on Day −17 and removal of the CIDR on Day −11 concurrently with 500µg of cloprostenol sodium. On Day −9, all heifers received either a new or once-used (used) CIDR and 100µg of gonadorelin acetate (GnRH, Parnell Inc.). Six days later (Day −3) CIDRs were removed, 1000µg of PGF was administered, and an oestrous detection patch applied (Estrotect, Rockway Inc.). At 72h after CIDR removal, 100µg of GnRH and AI using 3 different sires was performed. Pregnancy was determined by ultrasonography 31 days after AI. A subset of heifers (n=151) were examined on Day −9 and Day −3 by ultrasonography to assess ovulation to Day −9 GnRH. Data were analysed using GLIMMIX (SAS 9.4; SAS Institute Inc.). Presynch heifers had larger follicle diameter on Day −9 (12.7±0.3 vs. 10.4±0.3 mm; P<0.0001), greater ovulatory response (84.6%; 66/78 vs. 52.05%; 38/73; P<0.0001), greater oestrus expression (90.6%; 106/117 vs. 78.4%; 91/116; P=0.03), and expressed oestrus earlier (49.8±1.0 vs. 53.1±1.1 h; P=0.01) compared with controls. There was an interaction for treatment and CIDR on oestrous expression, whereas a lesser (P=0.003) percentage of control heifers with new CIDR showed oestrus compared with all other groups (Table 1). Heifers treated with a used P4 device tended (P=0.08) to have greater P/AI (52.1%; 61/117) than those with a new CIDR (40.5%; 47/116). In conclusion, presynchronization before initiation of a 6-day CO-Synch increased follicle diameter, ovulatory response, and oestrous expression, but did not affect fertility. The earlier onset of oestrus in Presynch heifers warrants further study on timing of AI. Table 1. Oestrous expression and pregnancy per AI (P/AI) in beef heifers with or without presynchronization and treated with a new or used CIDR Treatment CIDR Oestrus (%; n/n) Time of oestrus (h) P/AI (%; n/n) Control New 67.8a (40/59) 53.7±1.5a 33.9 (20/59) Used 89.5b (51/57) 52.7±1.6a 50.9 (29/57) Presynch New 94.7b (54/57) 50.9±1.4b 47.4 (27/57) Used 86.7b (52/60) 48.7±1.3b 53.3 (32/60) P-value Treatment 0.03 0.01 0.21 CIDR 0.62 0.18 0.08 Interaction 0.003 0.78 0.38 a,bValues with different superscripts differ (P < 0.05).

170 Embryonic loss and pregnancy rate in response to resynchronisation using oestradiol benzoate or injectable progesterone at 14 days after timed AI in Bos taurus×Bos indicus beef heifers

2020 ◽  
Vol 32 (2) ◽  
pp. 212
Author(s):  
C. C. Vieira ◽  
V. Buss ◽  
A. M. Oliveira ◽  
M. E. R. Costa ◽  
B. G. Freitas ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Pregnancy Rates ◽  
Beef Heifers ◽  
Graduate Studies ◽  
Oestradiol Benzoate ◽  
Embryonic Loss ◽  
Long Acting ◽  
Intravaginal Device

The effect of the exposure of pregnant cattle to oestradiol benzoate (EB) or injectable long-acting progesterone (iP4), as well as their use for early resynchronisation of non-pregnant (NP) females within the second week after timed AI (TAI), is yet to be assessed. This study aimed to assess the pregnancy rate and embryonic loss of cross-bred Bos taurus×Bos indicus beef heifers in response to an EB- or iP4-based resynchronisation strategy implemented 14 days after the first TAI. A total of 677 cyclic heifers (Braford and Brangus; 24 months old) were subjected to an EB/P4 synchronisation protocol for the first TAI (Day 0=TAI). On Day 14, all heifers received a new P4 intravaginal device (1g, Sincrogest, Ourofino Saúde Animal) and were randomly assigned to two groups: EB (1mg intramuscularly, Sincrodiol; n=339) or iP4 (75 mg intramuscularly, Sincrogest Injetável; n=338). On Day 22, P4 devices were removed and heifers underwent colour Doppler ultrasonography evaluation. Non-pregnant females were identified by the assessment of structural regression of the corpus luteum, as previously reported by our group. On Day 22, NP heifers received 1mg of oestradiol cipionate (SincroCP), 250µg of sodium cloprostenol (Sincrocio), and 200IU of equine chorionic gonadotrophin (SincroeCG), and they were inseminated on Day 24. On Day 22, the diameter of the preovulatory follicle (POF) was also recorded. Pregnancy was diagnosed via B-mode ultrasonography on Days 35-40 and 54. Statistical analyses were performed using SAS software (ver. 9.2, SAS Institute Inc.). The MIXED procedure was used to evaluate the POF diameter, and the GLIMMIX procedure was used to develop a multivariate model including treatment, breed, age, farm (1-7), and possible interactions of treatment with farm, breed, and age to evaluate the binomial data (pregnancy rates and embryonic loss). In NP heifers, the diameter of the POF on Day 22 did not differ (P>0.05) between the EB (9.77±0.25mm) and iP4 (9.92±0.22 mm) groups. Exposure to EB or iP4 on Day 14 post-TAI did not influence pregnancy rates as assessed on Day 22 (56.3% (191/339) vs. 60.1% (203/338)) and on Days 35-40 (49.6% (168/339) vs. 53.3% (180/338)). In addition, embryonic loss between Days 22 and 35-40 was not different between the EB (12%, 23/191) and iP4 (11.3%, 23/203) groups. On the other hand, pregnancy rates in heifers submitted to the second TAI were greater (P=0.01) in the EB group (45.95%, 68/148) than in the iP4 group (31.85%, 43/135). In conclusion, improved pregnancy rates can be achieved using 1mg of EB associated with a P4 intravaginal device on Day 14 of the resynchronisation protocol without affecting the first TAI pregnancy rates and embryonic loss. However, further investigation is warranted to assess the lower pregnancy rates obtained in response to the association of iP4 and P4 device. This research was supported by FAPESP (2015/10606-9; 2019/15514-6), Graduate Studies Support Program (PAPG-UNIPAMPA), and Ourofino Saúde Animal.

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