Numbers of antral follicles during follicular waves in cattle: Evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations

The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Follicular dynamics in zebu cattle

The aim of this study was to investigate the follicular dynamics during estrous cycle in Gir breed (Bos indicus) cows. Follicular growth and atresia during estrous cycle were evaluated using a portable ultrasound device. Luteal activity was evaluated by serum progesterone levels. Cycles with two (6.67%), three (60.00%), four (26.67%) and five (6.67%) follicular waves were observed. There was no difference (P>0.05) in dominant or subordinate follicles growth or atresia rates among follicular waves. The maximum diameter of the ovulatory follicle was higher than the diameter of the other dominant follicles in cycles with four waves, and higher than the diameter of the second dominant follicle in cycles with three waves (P<0.05). There was no difference (P>0.05) in estrous cycle length (21.11±1.76 and 22.25±1.71 days) or progesterone levels during diestrous (4.48±1.45 and 5.08±1.40 ng/mL) between cycles with three or four waves. Follicular dynamics in Gir cattle is characterized by a higher incidence of cycles with three or four waves, associated with a low persistence of the dominant follicle.

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

Follicular Development in Ewes During Natural and Prostaglandins Induced Estrous Cycle

The follicular development was evaluated in ovine females during natural and prostaglandin-F(2 alpha) (PG) induced estrous cycle. Ewes were randomly divided in two treatments (n=7/treatment): T1 with natural cycle and T2 synchronized with two injections of PG. From one day before PG injection until next ovulation, daily transrectal ultrasonography was done. All follicles >= 2 mm were assessed. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-3 waves). The maximum diameter of the largest follicle of the first wave was greater in T1 (5.83 +/- 0.31 mm) compared with T2 (5.0 +/- 0.1 mm; P<0.01), but there was no significant difference among the emergency day of largest follicle, during the growth phase of the follicular waves. The duration of the plateau phase in wave 2 differed between the two treatments (P<0.05) showing 0.83 +/- 0.31 and 1.83 +/- 0.17 d, for natural and synchronized treatment, respectively. Growth rate did not differ between treatments. Presence of new luteal tissue was detected on day 3 after ovulation. In conclusion, the follicular development was similar in female ovine during natural and PG induced estrous cycle.

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.

Follicular dynamics in Mangalarga mares.

Ovarian follicular activity was studied by ultrasonography during 17 oestrous cycles in 9 Mangalarga mares during the second half of the ovulatory season. Sixteen oestrous cycles were considered normal and one 3-wave cycle showing a prolonged luteal phase was considered atypical. Daily ultrasonographic examinations were performed and the compiled data on follicular dynamics were studied retrospectively. One major wave of follicular growth was observed in 13 of the 16 normal cycles (81.25%), whereas 2 major waves occurred in 3 cycles (18.75%). The mean (+/- s.d.) days of emergence of the primary wave of follicular development in cycles containing one or 2 waves were Day 6.0 +/- 2.3 and Day 11.0 +/- 1.0, respectively. The secondary wave of follicular development in 2-wave cycles emerged on Day 0.0 +/- 3.6. The day of wave divergence for primary waves of follicular development in cycles which exhibited one or 2 major waves were Day 12.2 +/- 3.5 and Day 17.3 +/- 3.0, respectively. Divergence of secondary waves occurred in only one of the 3 cycles which exhibited 2 major follicular waves (Day 7). The mean (+/- s.d.) maximum diameters of the dominant follicle in the primary wave of oestrous cycles exhibiting one and 2 major waves were 39.0 +/- 3.9 mm and 34.7 +/- 2.5 mm, respectively. The mean (+/- s.d.) maximum diameter of the dominant follicle present in the secondary wave was 34.3 +/- 11.0 mm. The mean (+/- s.d.) lengths of the interovulatory intervals for cycles containing one and 2 major waves were 19.4 +/- 2.2 and 23.3 +/- 2.5 days, respectively. These data indicate that most Mangalarga mares show one major follicular wave during the oestrous cycle but a small percentage of mares show 2 major waves.

Analysis of follicular events in owl monkeys (Aotus azarai infulatus) using B-mode and Doppler ultrasound

Ultrasound (B-mode) was used to analyze follicular events in 12 trained female owl monkeys (Aotus azarai infulatus). The animals were examined every 48 hours for over 90days to measure and map follicular growth in both ovaries and to measure (using Doppler velocimetry) local hemodynamic changes during the peri-ovulatory stage. There were 44 follicular growth events, each with two or three follicular waves, and a mean ± SEM interval between events of 17 ± 1.13 days. There were various hemodynamic changes during follicular growth; both vascular resistance index and pulsatility index decreased during the time when the follicle diameter peaked. Thus, both B-mode and Doppler ultrasound were useful for monitoring ovarian follicular events in owl monkeys. © 2013 Elsevier Inc.

Control of Buffalo Follicular Dynamics for Artificial Insemination, Superovulation and In Vitro Embryo Production

Currently, timed ovulation induction and timed artificial insemination (TAI) can be performed in buffalo using GnRH or estradiol plus progesterone/progestin (P4)-releasing devices and prostaglandin F-2 alpha (PGF(2 alpha)). The control of the emergence of follicular waves and of ovulation at predetermined times, without the need for estrus detection, has facilitated the management and improved the efficiency of AI programs in buffalo during the breeding and nonbreeding season. Multiple ovulations, embryo transfer, ovum collection and in vitro embryo production have been shown to be feasible in buffalo, although low efficiency and limited commercial application of these techniques have been documented as well. These results could be associated with low ovarian follicular pools, high levels of follicular atresia and failures of the oocyte to enter the oviduct after superstimulation of follicular growth. This review discusses a number of key points related to the manipulation of ovarian follicular growth to improve pregnancy rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in buffalo.

Reproductive cycles in Bos indicus cattle

Several studies using transrectal ovarian ultrasonic scanning in Bos taurus (B. taurus) cattle and more recently in Bos indicus (B. Indicus) females evaluated the reproductive cycles of heifers and cows under different conditions. In general, B. indicus cattle have more follicles and more follicular waves during the estrous cycle and ovulate from smaller follicles than B. taurus. Consequently B. indicus females have smaller corpora lutea and it is assumed circulating concentrations of estradiol and progesterone are also less. However, these findings may vary depending on the nutritional status and regimen in which the animals are managed. Moreover, there are significant differences between B. taurus and B. indicus regarding follicle size at the time of deviation of the dominant follicle. These differences in ovarian function between B. indicus and B. taurus, e.g. greater antral follicle population are, probably, the main reasons for the great success of in vitro embryo production programs in Zebu cattle, especially in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

Synchronization of ovulation in crossbred dairy heifers using gonadotrophin-releasing hormone agonist, prostaglandin F2a and human chorionic gonadotrophin or estradiol benzoate

Girolando (Gir x Holstein) is a very common dairy breed in Brazil because it combines the rusticity of Gir (Bos indicus) with the high milk yield of Holstein (Bos taurus). The ovarian follicular dynamics and hormonal treatments for synchronization of ovulation and timed artificial insemination were studied in Girolando heifers. The injection of a gonadotrophin-releasing hormone (GnRH) agonist was followed 6 or 7 days (d) later by prostaglandin F2a (PGF2a). Twenty-four hours after PGF2a injection either human chorionic gonadotropin (hCG, GPh-d6 and GPh-d7 groups) or estradiol benzoate (EB, GPE-d6 and GPE-d7 groups) was administered to synchronize ovulation and consequently allow timed artificial insemination (AI) 24 and 30 h after hCG and EB injection, respectively. Follicular dynamics in Girolando heifers was characterized by the predominance of three follicular waves (71.4%) with sizes of dominant follicles (10-13 mm) and corpus luteum (approximately 20 mm) similar to those for Bos indicus cattle. In the GnRH-PGF-hCG protocol, hCG administration induced earlier ovulation (67.4 h, P<0.01) compared to the control group (GnRH-PGF) and a better synchronization of ovulation, since most of it occurred within a period of 12 to 17 h. Pregnancy rate after timed AI was 42.8 (3/7, GPh-d6) to 50% (7/14, GPh-d7). In contrast, estradiol benzoate (GnRH-PGF-EB protocol) synchronized ovulation of only 5 of 11 heifers from the GPE-d7 group and of none (0/7) from the GPE-d6 group, which led to low pregnancy rates after timed AI (27.3 and 0%, respectively). However, since a small number of Girolando heifers was used to determine pregnancy rates in the present study, pregnancy rates should be confirmed with a larger number of animals.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

Dinâmica folicular e momento da ovulação em vacas não lactantes das raças Gir e Nelore durante duas estações do ano

Estudou-se o efeito da estação do ano na dinâmica folicular e no momento de ovulação utilizando-se 25 vacas não lactantes da raça Gir (10 no inverno e 15 no verão) e 15 vacas não lactantes da raça Nelore (sete no inverno e oito no verão), todas com ciclo estral normal. Após a detecção do estro, as vacas foram submetidas a exames ultra-sonográficos a cada seis horas com a finalidade de monitorar o momento de ovulação e, posteriormente, uma vez ao dia durante todo o ciclo estral, sendo monitorado um ciclo estral por animal e por estação do ano. As características foliculares não foram afetadas pela estação do ano e não diferiram entre as raças. Houve maior percentagem de ciclos com três (68,0 e 66,7%) e duas ondas foliculares (24,0 e 26,7%) nas raças Gir e Nelore, respectivamente. Os intervalos entre estros e entre ovulações foram menores para ciclos com duas ondas foliculares. A manifestação do estro ocorreu mais nas primeiras horas da manhã (57,3%) e no final da tarde e início da noite (28,0%). O momento de ovulação ocorreu 23,1±5,6 horas após a detecção do estro nas duas raças.

Synchronization of ovulation in crossbred dairy heifers using gonadotrophin-releasing hormone agonist, prostaglandin F2a and human chorionic gonadotrophin or estradiol benzoate

Girolando (Gir x Holstein) is a very common dairy breed in Brazil because it combines the rusticity of Gir (Bos indicus) with the high milk yield of Holstein (Bos taurus). The ovarian follicular dynamics and hormonal treatments for synchronization of ovulation and timed artificial insemination were studied in Girolando heifers. The injection of a gonadotrophin-releasing hormone (GnRH) agonist was followed 6 or 7 days (d) later by prostaglandin F2a (PGF2a). Twenty-four hours after PGF2a injection either human chorionic gonadotropin (hCG, GPh-d6 and GPh-d7 groups) or estradiol benzoate (EB, GPE-d6 and GPE-d7 groups) was administered to synchronize ovulation and consequently allow timed artificial insemination (AI) 24 and 30 h after hCG and EB injection, respectively. Follicular dynamics in Girolando heifers was characterized by the predominance of three follicular waves (71.4%) with sizes of dominant follicles (10-13 mm) and corpus luteum (approximately 20 mm) similar to those for Bos indicus cattle. In the GnRH-PGF-hCG protocol, hCG administration induced earlier ovulation (67.4 h, P<0.01) compared to the control group (GnRH-PGF) and a better synchronization of ovulation, since most of it occurred within a period of 12 to 17 h. Pregnancy rate after timed AI was 42.8 (3/7, GPh-d6) to 50% (7/14, GPh-d7). In contrast, estradiol benzoate (GnRH-PGF-EB protocol) synchronized ovulation of only 5 of 11 heifers from the GPE-d7 group and of none (0/7) from the GPE-d6 group, which led to low pregnancy rates after timed AI (27.3 and 0%, respectively). However, since a small number of Girolando heifers was used to determine pregnancy rates in the present study, pregnancy rates should be confirmed with a larger number of animals.

Perfil hormonal de Progesterona durante o ciclo Estral em novilhas Nelore confinadas com Diferentes Ondas de Crescimento Folicular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)