João Francisco Coelho Oliveira

Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by prostaglandins E2 and F2α

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 muM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P<0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 microM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P<0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE(2), PGF(2alpha) or AngII was present in the co-culture system with follicular cells (PGE(2) 77.4%, PGF(2alpha) 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE(2) or PGF(2alpha) produced by follicular cells.

Follicular development and steroid concentrations in cows with different levels of fertility raised under nutritional stress

The aim of the present study was to characterize ovarian follicular development and steroid concentrations during postpartum and the estrous cycle of Brangus Ibagé cows (3/8 Nelore + 5/8 Aberdeen Angus) with different levels of fertility. Cows were classified as having high or low fertility according to the calving interval (CI). The average CI of the herd from which cows used in this study were selected was 404.6+/-5.44 and 711.2+/-20.89 days for the high and low fertility groups, respectively. Four cows of high fertility and five cows of low fertility had calves removed between 70 and 100 days after parturition. Ovarian activity was monitored daily by ultrasound for 16 days after calf removal. Days to emergency of the first follicular wave after calf removal, number of follicles with diameter >9 mm, growth rate of largest follicle, maximum diameter of largest follicle, length (days) and number of follicular waves were recorded. During this period, blood was collected daily for measurements of serum progesterone (P(4)) and estradiol (E(2)) concentrations. In another experiment, ovarian activity and P(4) and E(2) concentrations were examined during estrous cycle in five cows of high fertility and four cows of low fertility. Ovarian activity and steroid concentrations were assessed from the day prior to estrus to the 15th day of the estrous cycle (estrus = day 0). In postpartum cows of high fertility, the total number of follicles >5mm and the maximum diameter of the largest follicle were higher than in cows of low fertility (P < 0.05). Concentrations of P(4) and E(2) did not differ between groups in the postpartum cows. However, E(2) increased 5 days after calf removal (around 90 days of postpartum) in the high fertility group, followed by an increase in P(4) with average values indicating ovulation around 100 days postpartum. In cycling cows, the profile of follicular development was similar between cows of high and low fertility. There was no difference between groups for number of follicles >5mm, but the day effect was significant (P < 0.01). Plasma concentrations of P(4) and E(2) were similar in both groups. These data suggest that cows, from a population raised in the same environment have different fertility as a consequence of individual physiological characteristics.

Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by PGE2 and PGF2 1

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 muM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P<0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 microM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P<0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE(2), PGF(2alpha) or AngII was present in the co-culture system with follicular cells (PGE(2) 77.4%, PGF(2alpha) 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE(2) or PGF(2alpha) produced by follicular cells.

Expression of receptors for BMP15 is differentially regulated in dominant and subordinate follicles during follicle deviation in cattle

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P<0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P<0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.

Effects of estradiol and progestins on follicular regression before, during, and after follicular deviation in postpartum beef cows

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60-90 d postpartum, given 125 microg cloprostenol on two occassions, 12h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24h until Day 7 (Day 0=treatment). When the largest follicle reached a designated diameter of 5-7, 8-10 or >10mm, cows were randomly allocated to receive 2mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100mg of progesterone (P(4)) given im, or an intravaginal device containing P(4) (3 x 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P(4) delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.

Assessment of bovine spermatozoa viability using different cooling protocols prior to cryopreservation

The aim of our study was to evaluate the effect of different cooling rates on the post-thawing quality of bovine spermatozoa. Ejaculated semen from a 24-month-old Jersey bull was collected using an artificial vagina and diluted in a commercial extender to evaluate spermatozoan concentration and motility subjectively before cooling and freezing and after thawing. Straws were allocated to four cooling curves: rapid (RD), semi-rapid (SRD), semi-slow (SSLW) and slow (SLW). The temperature was decreased from 25°C to 4°C in 10, 50, 110 and 135 min, which represents a cooling rate of 2.06, 0.40, 0.18 and 0.15°C/min, respectively. Then straws were frozen and stored at −196°C. After thawing, one aliquot of each straw was used for evaluation. Spermatozoan integrity and mitochondrial function were evaluated using a combination of fluorescent probes containing 100 mg/mL FITC-PSA, 0.5 µg/mL PI and 153 µM JC-1. At the end of cooling, spermatozoan motility did not differ among RD (63.3%), SRD (66.7%), SSLW (66.7%) and SLW (80.0%). However, normal spermatozoan morphology was lower in SRD (84.8%) compared to RD (91.7%), SSLW (91.7%) and SLW (90.3%) (P<0.05). In thawed semen, spermatozoan motility and normal morphology did not differ among RD (40.0%; 88.8%), SRD (43.3%; 82.5%), SSLW (40.0%; 87.2%) and SLW (36.7%; 88.0%). The percentage of damaged spermatozoa, including plasma and acrosome membrane damage and low mitochondrial potential, was higher in RD compared to the others (P<0.05). In conclusion, a rapid cooling curve is detrimental to the spermatozoa and affects the post-thaw spermatozoan integrity of bovine frozen semen.

Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos

The aim of this study was to evaluate the effect of protein kinase C (PK-C) on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC) and denuded oocytes (DO), randomly allotted to 6 treatments (T) based on the presence of an activator of PK-C (PMA) (T1 and T2), or a phorbol ester unable to activate PK-C (4aPDD-control) (T3 and T4) or a basic culture medium (T5 and T6), were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD) was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was dependent of cumulus cells to affect the progression to the stages of metaphase I (MI) and metaphase II (MII) at 12 and 18 hours of culture. At 9 and 22 hours, no difference among groups was detected. PK-C accelerates the meiotic resumption independently of the somatic cells but depends on cumulus cells for the progression to the stages of MI and MII.

Identification of molecular markers on bovine chromosome 18 associated to calving interval in a Brangus-Ibagé cattle herd

In the detection phase of a bovine marker assisted selection program, this paper investigated the genetic variability of three microsatellites on the chromosome 18 (BTA 18). The possible associations between genotypes or alleles of these markers versus weight at first calving and a lifetime calving interval (as indicators of reproductive performance) were evaluated in a beef cattle herd (5/8 Aberdeen Angus x 3/8 Nelore). Eleven alleles were detected in TGLA227 and ILSTS002 and three in BMS3004, the most frequent being TGLA227*79, ILSTS002*133, ILSTS002*135 and BMS3004*129. Polymorphic information content ranged from 0.41 to 0.84, while heterozygosity ranged from 49% to 86%, with an average value of 77%. The association analyses performed between genotype classes for the genetic markers versus weight at first calving indicated no significant result. Also, no correlation was observed between calving interval (CI) and TGLA227 genotypes. However, positive associations were detected between ILSTS002 and BMS3004 and CI. Animals carrying at least one ILSTS002*135 allele presented a CI about 39 days longer than the individuals with other genotypes; animals heterozygous for BMS3004 presented a CI about 35 days shorter than the homozygous. On these grounds, it can be concluded that these markers can be useful as an aid to fertility selection, in this herd.

Associação entre polimorfismos do gene IGF-IR e características produtivas e reprodutivas em fêmeas bovinas da raça Holandesa

The association between single-strand conformation polymorphism (SSCP) in the gene of insulin-like growth factor-I receptor (IGF-IR) and age at first calving (AFC), calving interval (CI), lactation length (LL), and milk yield (MY) was studied using 106 graded Holstein females. The polimerase chain reaction (PCR) with specific initiating oligonucleotides, resulted an amplified fragment of 335pb. The population genotypes frequencies were 82.1% and 17.9%, for AA and AB genotypes, respectively. The frequency of A allele was 0.91 and 0.09 of B allele. No association between the identified polymorphism and AFC, CI, and MY was observed. The LL was positively associated (P<0.05) with the absence of B allele. Animals carrying the AA genotype presented a longer lactation period.

Interaction between estrogen receptor and retinol-binding protein-4 polymorphisms as a tool for the selection of prolific pigs

The aim of the present study was to investigate the association of the estrogen receptor (ER-PvuII) and retinol-binding protein 4 (RBP4-MspI) gene polymorphisms and their interactions with prolificacy in a commercial synthetic pig line reared in Brazil. A total of 10,374 piglet records from 218 sows and 817 litters were used for litter size analysis. Only females with three or four farrowings were included in the analysis. The mean litter size ranged from 5.0 to 19.5 piglets. DNA was extracted from leukocytes by a standard method, and ER-PvuII and RBP4-MspI polymorphisms were characterized by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The association between alleles or genotypes and reproductive performance was analyzed using a general linear model including the interaction between the ER-PvuII and RBP4-MspI genotypes. For the ER-PvuII gene, the allele frequencies of allele A and allele B were 0.56 and 0.44, respectively. For the RBP4-MspI gene, the frequencies of alleles A1 and A2 were 0.29 and 0.71, respectively. The total number of piglets born (TNB), born alive (NBA), or number of mummies and stillborn piglets (NMUM and NSB) per litter did not differ between the various ER-PvuII and RBP4-MspI genotypes. However, when the ER-PvuII and RBP4-MspI genotypes were considered together in each sow, TNB and NBA were 1.4 (p = 0.0026) and 0.9 (p = 0.019) higher in AA/A1 and AB/A1 animals, respectively, than in AA/A2 and BB/A1 animals. Likewise, TNB and NBA were 0.9 (p = 0.0258) and 0.8 (p = 0.0168) higher in BB/A2 and AB/A2 sows, respectively, than in AA/A2 and BB/A1 animals, but no difference was observed compared to AA/A1 and AB/A1 animals. The results showed larger litter sizes (TNB and NBA) for sows carrying the ER-PvuII allele A and the RBP4-MspI genotype A1, and for animals carrying the ER-PvuII allele B and the RBP4-MspI genotype A2. In conclusion, the interaction between genotypes ER-PvuII and RBP4-MspI is more efficient in the selection of prolific sows than each one of these molecular markers alone.