Cisto ovariano em vacas de leite: incidência, resposta à aplicação de GnRH e desempenho reprodutivo

A incidência de cistos ovarianos, a resposta ao tratamento com GnRH e os efeitos da ocorrência de cisto no desempenho reprodutivo e na taxa de descarte foram determinados em vacas lactantes da raça Holandesa. Vacas lactantes (n=333) foram avaliadas semanalmente por ultrassonografia a partir da quarta semana pós-parto, visando à detecção de corpos lúteos (CL) e de folículos ovarianos maiores que 10mm. Na sétima semana pós-parto, as vacas foram classificadas: em ciclando (n=248; presença de CL em um dos exames ultrassonográficos); em anestro (n=54; ausência de CL e de folículos >25mm) e com cisto (n=31; ausência de CL e presença de estruturas >25mm), quando foram distribuídas em: grupo-controle (n=16; sem tratamento) e grupo-tratamento (n=15; vacas que receberam uma aplicação de GnRH). A taxa de cura foi de 60,0% no grupo das vacas tratadas e de 87,5% no grupo-controle. As vacas com cistos apresentaram maior intervalo parto-primeira inseminação artificial (P<0,05; 91,4±8,3 vs. 77,8±2,5), maior número de serviços por concepção (P<0,05; 4,4±1,2 vs. 3,6±1,5), maior intervalo parto-concepção (P<0,05; 214,8±25,9 vs. 174,9±7,7) e maior taxa de descarte (P<0,05; 41,2 vs. 21,8%) do que as vacas ciclando. Vacas diagnosticadas com cisto na sétima semana pós-parto apresentaram recuperação espontânea, embora o intervalo parto-concepção e a taxa de descarte tenham sido maiores para essas vacas.

Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries - partial results

Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36 degrees C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5 degrees C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 mu l of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

Morphologic and quantitative evaluation of preantral follicles of buffalo (Bubalus bubalis) in different reproductive phases

The objectives of the present study were to estimate the population of the normal and degenerated preantral follicles of buffaloes in different reproductive phases and to classify the different types of degeneration of ovarian preantral follicles. For this, 18 ovaries were divided in three groups: prepubertal, non pregnat and pregnant adult females. The ovaries were collected from a slaughterhouse (Frigol - Brazil) and processed for classic histological examination. The follicular population was estimated according to Cahill et al. (1979) and calculated according to Gougeon & Chainy (1987). Young buffaloes, pregnant and non pregnat adults presented an average number of 15.5, 3.7 and 8.9 preantral follicles with normal morphology and type I (the degeneration was observed just in oocyte) and II (oocyte and granulosa cells were degenerated) degeneration, respectively. This experiment demonstrated that the proportions of primordial, primary and secondary follicles were affected by reproductive phase. A significant difference was observed considering the proportions of preantral follicles with normal morphology and Type I degeneration in all reproductive phases evaluated. The primary follicles presented more percentage of Type I degeneration that the others follicles and the primordial and primary follicles presented more percentage of Type II degeneration in three reproductive phases evaluated.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation (3) administration of EPE twice daily in successively decreasing doses. In the first experiment. 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of greater than or equal to35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation. or two doses of a GnRH-a agonist upon detection of a follicle greater than or equal to35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles greater than or equal to35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF(2gamma) and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF(2alpha), but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles :35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose. improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates. (C) 2002 Elsevier B.V. All rights reserved.

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6).Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection. (C) 2001 by Elsevier B.V.

Effect of eFSH on ovarian cyclicity and embryo production of mares in spring transitional phase

The use of equine FSH (eFSH) for inducing follicular development and ovulation in transitional mares was evaluated. Twenty-seven mares, from 3 to 15 years of age, were examined during the months of August and September 2004, in Brazil. Ultrasound evaluations were performed during 2 weeks before the start of the experiment to confirm transitional characteristics (no follicles larger than 25 mm and no corpus luteum [CL] present). After this period, as the mares obtained a follicle of at least 25 mm, they were assigned to one of two groups: (1) control group, untreated; (2) treated with 12.5 mg eFSH, 2 times per day, until at least half of all follicles larger than 30 mm had reached 35 mm. Follicular activity of all mares was monitored. When most of the follicles from treated mares and a single follicle from control mares acquired a preovulatory size ( : 35 mm), 2,500 IU human chorionic gonadotropin (hCG) was administered IV to induce ovulation. After hCG administration, the mares were inseminated with fresh semen every other day until ovulation. Ultrasound examinations continued until detection of the last ovulation, and embryo recovery was performed 7 to 8 days after ovulation. The mares of the treated group reached the first preovulatoiy follicle (4.1 +/- 1.0 vs 14.9 +/- 10.8 days) and ovulated before untreated mares (6.6 +/- 1.2 vs 18.0 +/- 11.1 days; P <.05). All mares were treated with prostaglandin F-2 alpha (PGF(2 alpha)), on the day of embryo flushing. Three superovulated mares did not cycle immediately after PGF(2 alpha), treatment, and consequently had a longer interovulatory interval (22.4 vs 10.9 days, P < 0.05). The mean period of treatment was 4.79 1.07 days and 85.71% of mares had multiple ovulations. The number of ovulations (5.6 vs 1.0) and embryos (2.0 vs 0.7) per mare were higher (P < 0.05) for treated mares than control mares. In conclusion, treatment with eFSH was effective in hastening the onset of the breeding season, inducing multiple ovulations, and increasing embryo production in transitional mares. This is the first report showing the use of FSH treatment to recover embryos from the first cycle of the year.

Expression of fibroblast growth factor-8 and its cognate receptors, fibroblast growth factor receptor (FGFR)-3c and-4, in fetal bovine preantral follicles

Paracrine cell signaling is thought to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family have been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR-3c and FGFR-4) are expressed in bovine preantral follicles. Reverse transcription-polymerase chain reaction was used to amplify bovine FGF-8, FGFR-3c, and FGFR-4 from preantral follicle samples and a variety of fetal and adult tissues. All three genes were widely expressed in fetal tissues, with a restricted expression pattern in adult tissues. FGF-8 and FGFR-3c were expressed in secondary follicles in 70% of fetuses examined, whereas FGFR-4 expression was significantly less frequent (20%). FGFR-3c expression frequency was significantly lower in primordial compared to secondary follicles, and FGF-8 expression showed a similar trend. FGFR-4 was only observed when all follicle classes of an individual were expressing both FGF-8 and FGFR-3c. We conclude that FGF-8 and its receptors are expressed in preantral follicles in a developmentally regulated manner. (C) 2005 Wiley-Liss, Inc.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 213, in bovine follicles

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and-4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.

Ovarian follicular dynamics in Nelore breed (Bos indicus) cattle

The most common beef cattle raised in Brazil is the Nelore breed (Bos indicus). Information obtained by ultrasonography on follicular growth in Bos taurus cattle has been accumulating rapidly. However, there are few publications to date on follicular development in Bos indicus breeds. The follicular dynamics in Nelore heifers and cows during natural or prostaglandin (PG)-induced estrous cycle were studied. From the detection of estrus onward, all animals were examined daily by ultrasonography for one (n=35) or two (n=10) consecutive estrous cycles. The follicular dynamic in Nelore cattle was characterized by the predominance of 2 follicular waves in the cows (83.3%, n=18, P<0.05) and 3 waves in the heifers (64.7%, n=16, P<0.05). Most of the cattle observed over 2 consecutive estrous cycles presented the same pattern of follicular waves in the first and second cycle, and only 30% showed variation in the number of waves from one cycle to the other. Most of the follicular parameters analyzed were not affected by PG treatment or age but were altered by follicular waves. Consequently, data on cows and heifers were combined according to the number of follicular waves. The ovulatory follicle was larger than the other dominant follicles (P<0.05), and the ovulatory wave was shorter than the preceding waves (P<0.05). The interovulatory interval was longer in animals showing 3 waves than those exhibiting 2 waves (P<0.05). Maximum diameter of the dominant follicle (around 11 mm) and of the corpus luteum (CL, approximately 17 mm) were smatter than those reported for European breeds. In conclusion, the results demonstrate that although the dominant follicle and corpus luteum are smaller than in European breeds, the follicular dynamics in Nelore cattle were similar to those observed in European breeds and were characterized by 2 or 3 follicular waves for cows and heifers, respectively, during the natural or prostaglandin-induced estrous cycle. (C) 1997 by Elsevier B.V.

Effects of dominant follicle aspiration and treatment with recombinant bovine somatotropin (BST) on ovarian follicular development in Nelore (Bos indicus) heifers

Follicle ablation has been recognized as an efficient method of follicular wave synchronization. Treatment with recombinant bovine somatotropin (BST) has been shown to enhance follicular development in <(Bos taurus)under bar>. This experiment assessed the effects of these treatments in Nelore (<(B. indicus)under bar>) heifers. Eight cycling Nelore heifers were randomly assigned to 3 different treatments. on Day 2 of a synchronized cycle (Day 0 = day of ovulation), heifers assigned to Treatments 1 and 2 received 2 mL of saline, whereas heifers assigned to Treatment 3 received 320 mg of BST. on Day 5, the first-wave dominant follicle was ablated by ultrasoundguided transvaginal aspiration in heifers in Treatments 2 and 3, and all heifers received an injection of prostaglandin on Day 11. Aspiration of the dominant follicle advanced and synchronized (P < 0.05) the day of second-wave emergence (6.9 +/- 0.1 vs. 8.4 +/- 0.4) and the day of the pre-wave FSH peak (6.0 +/- 0.0 vs. 6.9 +/- 0.4), and increased FSH peak concentrations (381 +/- 21 vs. 292 +/- 30; pg/mL; P < 0.01). Recombinant bovine somatotropin treatment caused a two-fold increase in plasma insulin-like growth factor-I (IGF-I) concentrations (P < 0.001) and resulted in a 36% increase in the number of small follicles (<5 mm; P < 0.001) compared with saline-treated heifers. In summary, in agreement with previous reports on <(B. taurus)under bar>, dominant follicle aspiration synchronized ovarian follicular development, and BST treatment increased peripheral concentrations of IGF-I in Nelore heifers. Recombinant bovine somatotropin also increased the number of small follicles, but this response appeared to be inferior to that reported for <(B. taurus)under bar>. (C) 2000 by Elsevier B.V.

Ovarian follicle diameter at timed insemination and estrous response influence likelihood of ovulation and pregnancy after estrous synchronization with progesterone or progestin-based protocols in suckled Bos indicus cows

The objectives of the present study were to evaluate factors associated with estrous synchronization responses and pregnancy per insemination (P/AI) in Bos indicus beef cows submitted to progesterone-based fixed-time artificial insemination (FTAI) protocols. A total of 2388 cows (1869 Nellore and 519 crossbred Nellore x Angus) from 10 commercial farms were evaluated to determine the relationships among breed, body condition score (BCS) on the first day of the FTAI protocol, the occurrence of estrus between progesterone device removal and FTAI and diameter of largest ovarian follicle (LF) at FTAI on estrous synchronization responses and P/AI. Cows (n=412 primiparous; 1976 multiparous) received an intravaginal device containing progesterone or an ear implant containing norgestomet (a progestin), and an injection of estradiol at the beginning of the estrous synchronization protocol. Body condition was scored using a 1-5 scale on the first day of the FTAI protocol and at 30-60 days postpartum. Females received 300IU of equine chorionic gonadotropin (eCG) and PGF(2 alpha) on the day the progesterone device/implant was removed and were inseminated 48-60h later. At insemination, cows (n=2388) were submitted to an ultrasonographic exam to determine the diameter of the LF. Follicles were classified into four categories based on mean and standard deviation (SD) of the LF (LF1 = two SD below the mean; LF2 = mean minus one SD; LF3 = mean plus one SD; LF4 = two SD above the mean). Ovulation rate was determined in a subset of cows (n=813) by three consecutive ultrasonographic exams: (1) at time of progesterone device/implant removal, (2) at time of FTAI and (3) 48 h after FTAI. Ovulation was defined as the disappearance of a large follicle (>= 8.0 mm) that was previously recorded. Estrus was determined in a subset of the cows (n = 445) by the activation of a detection of estrous patch placed on the tail head on the day of progesterone device/implant removal. Pregnancy was diagnosed 30 days after FTAI. Pregnancy was influenced (P = 0.001) by follicle diameter [LF1 = 27.5% (81/295), LF2 = 46.6% (328/705), LF3 = 57.9% (647/1118), LF4 = 63.3% (171/270)] and the occurrence of estrus [estrus = 67.7% (174/257) and no estrus = 36.2% (68/188)]. Follicle diameter at FTAI influenced ovulation rate [LF1 = 42.5% (34/80), LF2 = 73.9% (161/218), LF3 = 95.8% (407/425), LF4 = 97.8% (88/90)], the occurrence of estrus [LF1 = 54.8% (51/93), LF2 = 33.6% (43/128), LF3 = 68.9% (126/183), LF4 = 90.2% (37/41)] and P/AI among cows that had ovulations [LF1 =32.4% (11/34), LF2 = 50.3% (81/161), LF3 = 60.0% (244/407), LF4 = 68.2% (60/88)]. Improving estrous responses between progesterone device withdrawal and FTAI and increasing the diameter of the LF at FTAI may be important aspects to achieve improved estrous synchronization responses and P/AI following progesterone/progestin and estradiol based FTAI protocols in suckled Bos indicus cows. (C) 2010 Elsevier B.V. All rights reserved.

Inhibin B and the ovarian follicular reserve

Inhibins are dimeric glycoproteins composed of an α-subunit and a βA-subunit (inhibin A) or βB-subunit (inhibin B), which inhibits pituitary gonadotropin secretion of FSH. The inhibin B is a product of the cohort of antral follicles. The ovarian follicle number decrease steadily as a function of increasing age, with consequent falls in the levels of inhibin B and increase of FSH levels. It is sufficient to maintain ovulatory function and continued secretion of estradiol. Elevated FSH levels seem to occur late in the sequence of events associated with ovarian failure. The inhibin B, produced by granulosa cells, is the earliest marker of the decline in ovarian follicular reserve across reproductive aging.

Associated and independent comparisons between the two largest follicles preceding follicle deviation in cattle

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4-to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.