Follicle Wave Research Articles

Hormonal mechanisms regulating follicular wave dynamics I: Comparison of follicle growth profiles under different physiological conditions in heifers

Follicle diameter deviation has been identified as the pivotal morphological manifestation of follicle selection, however, many of the hormonal mechanisms leading to this key event and regulating variation between individual follicular waves remain undefined. This study compared circulating FSH, LH, and P4 with the follicular dynamics during three different physiological conditions. We hypothesized that these end-points would: 1) be similar for a spontaneous wave 2 vs one induced by follicular aspiration, 2) but would differ between wave 1 and 2, and 3) between conventional (F2 > 7.0 mm at deviation) vs undersized (F2 < 7.0 mm) deviations in either wave. Holstein dairy heifers (N = 24) were studied daily during an interovulatory interval. All heifers were evaluated during wave 1 and randomized 6 days after ovulation into an induced wave 2 and a spontaneous wave 2. Values were normalized to the day of expected diameter deviation (day 0) and compared for day −2 to 0 and 0 to 2. Hypothesis 1 was supported that an induced wave 2 from ablation of follicles of wave 1 and spontaneous wave 2 have similar follicle dynamics. However, the peak FSH surge was more prominent at emergence of an induced wave 2 (P < 0.003). Hypothesis 2 was supported that waves 1 and 2 differ in follicle and hormone dynamics. Circulating P4 was lower and LH was greater (P < 0.01) with no difference in diameter of F1 but with a greater (P < 0.01) diameter of F2 on day 0 in wave 1 (7.3 ± 0.2 mm) than in wave 2 (6.6 ± 0.2 mm). Differences between waves were not found when each follicular wave was categorized into conventional vs undersized deviation and analyzed separately. Hypothesis 3 was supported as there were differences in circulating hormones between conventional and undersized deviations. Growth rate of F2 differed (P < 0.0005) during days −2 to 0 (conventional, 2.6 ± 0.2 mm/2d; undersized, 1.4 ± 0.3 mm/2d). However, circulating FSH and P4 concentration on days −1 and 0 tended to be greater (P < 0.06) in undersized than conventional deviations. In conclusion, the effect of different hormonal conditions on follicle dynamics was observed for F2 and not for F1. Furthermore, understanding the physiology that produces conventional vs undersized deviations is crucial since these categories explained most differences in follicular dynamics and circulating FSH observed in these different physiological conditions. In addition, future studies of wave 2 may be facilitated by using an induced wave 2 since it was similar to a spontaneous wave 2.

Spontaneous switching of future dominance to a smaller follicle: commonality among monovular species.

Selection of a dominant follicle from a wave of follicles is manifested by diameter deviation between future dominant (F1) and largest subordinate (F2) follicles. On day -1 or 0 (day 0=beginning of deviation), growth rate of F1 continues and growth rate of F2 decreases. Deviation occurs during the decline in the wave-stimulating FSH surge when F1 reaches means of 8.5, 10.5, and 22.5 mm in heifers, women, and mares, respectively. Diameter of F1 at the FSH peak vs at deviation is proportionally similar among these monovular species. In conventional deviation, F1 usually emerges first. In F1,F2-switched deviation, F2 is usually first to emerge and to reach a diameter characteristic of deviation. On day -1 or 0, the larger F2 and the smaller F1 switch so that the formerly larger F2 becomes subordinate and the formerly smaller F1 becomes dominant. In heifers and mares, the profile and prominence of the FSH surge are similar between deviation classes. Surge location relative to deviation differs so that the surge ends earlier in switched deviation. When the larger F2 reaches a diameter characteristic of deviation, FSH concentration is too low for continued growth of F2. The decrease in FSH ceases (heifers) or increases (mares) presumably from a decrease in FSH inhibitors; therefore, F1 continues to grow and becomes dominant. The frequency of switched deviation (e.g., 16 to 37% among species) can be problematic in follicle-selection research. Switching is a natural model for study of the interplay between follicles and FSH.

Follicle growth and endocrine dynamics in women with spontaneous luteinized unruptured follicles versus ovulation.

Do growth patterns and endocrine profiles differ between ovulatory follicles (OvFs) and luteinized unruptured follicles (LUFs) in women? Growth rates, diameters and associated endocrine profiles differed between OvFs and LUFs in unstimulated cycles. Two-three waves of antral follicles develop during the menstrual cycle in ovulatory women of reproductive age, with the second or third wave terminating in ovulation. In contrast, some women can develop LUFs, where a preovulatory follicle fails to rupture and there is subsequent luteinization of the follicle wall. However, no study has compared OvFs and LUFs in unstimulated cycles. This retrospective observational study was conducted in 56 healthy women of reproductive age (range: 19-41 years) and with a history of regular menstrual cycles. Participants who met inclusion criteria were enrolled, as previously reported. Daily transvaginal ultrasonography was performed for one interovulatory interval (IOI) to measure the diameters of all follicles >2 mm. Blood samples were collected every 3 days during the IOI to measure serum concentrations of FSH, LH, estradiol and progesterone. The interval from emergence to deviation (i.e. follicle selection) was shorter (P < 0.05) for LUFs compared to OvFs. However, the intervals from emergence to maximum diameter and deviation to maximum diameter were longer (P < 0.05) for LUFs compared to OvFs. Follicle deviation in LUFs occurred at a larger diameter (P < 0.05) compared to OvFs, and LUFs grew to larger (P < 0.0001) diameters compared to OvFs. Moreover, LUFs grew faster (P < 0.05) from emergence to deviation and from deviation to maximum diameter, compared to OvFs. LUFs were associated with low (P < 0.05) systemic LH levels at emergence and maximum diameter compared to OvFs. LUFs were also associated with low (P < 0.05) systemic FSH and high (P < 0.05) systemic progesterone at deviation and maximum diameter, respectively. Estradiol was higher (P < 0.05) at deviation and lower (P < 0.05) at maximum diameter for LUFs compared to OvFs. A 3-day interval of blood sampling for hormonal analyses was conducted, as a more frequent sampling interval was not considered acceptable by the study volunteers. A 3-day sampling interval did not allow characterization of acute changes in hormone production during the IOI. In addition, study visits were less frequent when LUFs persisted long after the expected day of the second ovulation of the IOI. Information about the growth and endocrine dynamics of OvFs and LUFs developing in unstimulated cycles in women may be applied to the early detection of LUF-associated anovulatory infertility and clinical management of women with this condition. No external funding sources were used for this study. The authors have no conflicts of interest in publishing this manuscript. ClinicalTrials.gov Identifier: NCT01389141.

Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle.

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. Luteal function in cattle has been studied in detail, and prostaglandin F2α has been used for several years for the elective induction of luteal regression. More recently, follicle wave dynamics has been studied and protocols designed to induce follicular wave emergence and ovulation have reduced, and even eliminated, the need for estrus detection. The addition of progestin-releasing devices, estradiol, GnRH and equine chorionic gonadotropin (eCG) have provided opportunities for fixed-time AI (FTAI) and possibilities for increased pregnancy rates. In embryo transfer programs, these same treatments have eliminated the need for estrus detection, permitting fixed-time embryo transfer and the initiation of superstimulatory treatments without regard to day of the estrous cycle. Collectively, new treatment protocols have facilitated the application of assisted reproductive technologies, and this is especially true in South America. Over the last 20 years, the use of AI in South America has increased, due largely to the use of FTAI. There has been more than a 10-fold increase in the use of FTAI in Brazil with more than 11 million treatments in 2016, representing 85% of all AI. Similar trends are occurring in Argentina and Uruguay. Production of in vivo-derived (IVD) embryos has remained relatively stable over the years, but in vitro embryo production (IVP) has increased dramatically over the past 10 to 15 years, especially in Brazil where more than 300,000 IVP embryos were produced in 2010. World-wide, more than 666,000 bovine IVP embryos were produced in 2016, of which more than 57% were produced in South America. The use of assisted reproductive technologies has facilitated the dissemination of improved genetics and increased reproductive performance; other South American countries are now following suit.

OvSynch program, its modifications and alternative hormonal programs in cow reproduction

The OvSynch program was formulated over 20 years ago (it consists in administering gonadoliberin on the day of starting the procedure, then injecting prostaglandin 7 days after the GnRH initial injection, and administration of GnRH two days after applying PGF2α. The procedure ends in the insemination of a female 16–20 hours after administering the final dose of the hormone) and it is commonly applied in cow reproduction management protocols. It has evolved into numerous modifications which has boosted its effectiveness. They have consisted in replacing GnRH with hCG or pLH, diversifying the time of the final injection of GnRH, which follows the prostaglandin injection, and postponing insemination after the final GnRH dose, as well as changing the dosage of the aforementioned hormones. To expedite the Ovsynch protocol, so-called PreSynch was used, which involved administering two prostaglandin injections with a 14 day interval before applying the initial GnRH dose in protocol OvSynch. The interval between the second prostaglandin injection in the PreSynch program varied (PreSynch-14, PreSynch-12, PreSynch-11, PreSynch-11 and PreSynch). The pregnancy rate can be also improved by introducing GnRH injection before starting the OvSynch program, administered 7, 6 or 4 days before the initial GnRH injection in OvSynch (G7G, G6G, G4G). Higher pregnancy rates, in comparison to the PreSynch-OvSynch program, can be obtained by applying the DoubleOvsynch protocol. In this protocol two OvSynch programs are administered in a 7-day interval. Lately, alternative programs such as DoubleSynch and EstraDoubleSynch have emerged. The DoubleSynch protocol consists in administering PGF2α in day 0, GnRH on the second day, another PGF application on the 9th day, and the second GnRH dose on the 11th day. Artificial insemination is performed in oriented time (TAI). This solution is used in order to achieve a higher percentage of pregnant cows in comparison to the PreSynch+OvSynch protocol. It also enables synchronisation of heat and ovulation, both after the first and the second hormonal treatment of GnRH. In EstraDoubleSynch protocol the second injection of GnRH at the end of the DoubleSynch program is replaced by estradiol benzoate (BE) administration on the 10th day, in order to acquire a higher level of follicle waves synchronisation and ovulation rate. Those programs applied in cow reproduction management can positively influence fertility and boost cow breeding profitability..

Programs for fixed-time artificial insemination in South American beef cattle.

Fixed-time artificial insemination (FTAI) has been widely applied in South America within the last 20 years for the genetic improvement of commercial beef herds. Most FTAI treatments for beef cattle used in South America are based on the use of progesterone (P4) releasing devices and estradiol to synchronize follicle wave emergence, with pregnancies per AI (P/AI) ranging from 40 to 60%. More recent protocols focusing on extending the interval from device removal to FTAI (i.e. increasing the growing period of the ovulatory follicle) have been reported to improve P/AI in beef cattle. These new protocols and the more traditional FTAI protocols have also been adapted for use with sexed-sorted semen with acceptable P/AI in beef cattle. Finally, color-flow Doppler ultrasonography has been incorporated recently to determine the vascularity of the CL and thereby detect pregnancy as early as Day 22 after the first AI for re- synchronization of ovulation for a second FTAI in non- pregnant animals. In summary, FTAI protocols have facilitated the widespread application of AI in South American beef cattle by allowing for the insemination and re-insemination of herds during a defined breeding season, without the necessity of clean up bulls to achieve high pregnancy rates.

133 In Vitro Embryo Production from Oocytes Collected from Non-Supertimulated Wood Bison (Bison bison athabascae) Following Maturation In Vitro Using Portable Incubators

This study was done to determine the feasibility of in vitro embryo production in wood bison during the anovulatory season, without ovarian superstimulation or follicle wave synchronization, to simulate collection conditions in a wild or field setting. The experiment provided the opportunity to compare embryo development using 2 different maturation media and incubator systems. The cumulus-oocyte complexes (COC) were collected by transvaginal ultrasound-guided follicle aspiration during May from non-superstimulated bison. Compact COC were allocated to 2 groups and matured in standard maturation medium using a portable gassed incubator, or in commercial medium using a portable non-gassed incubator. In the former (Standard), the COC were placed in a round-bottomed tube containing TCM-199 medium with 5% calf serum, 5 μg mL−1 LH, 0.5 μg mL−1 FSH, and 0.05 μg mL−1 gentamicin, and the tube was placed in a portable incubator with 5% CO2. In the latter (Commercial), COC were placed in a round-bottom tube containing the commercial medium (Boviteq, Saint-Hyacinthe, QC, Canada), and placed in a portable incubator without CO2. After 24 h of maturation, oocytes were fertilized in vitro (Day 0) in Brackett-Oliphant medium at 38.5°C in a conventional incubator with 5% CO2 humidified atmosphere. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3 and embryo development was recorded on Day 7. Cleavage and transferable embryo rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-squared test. No difference in cleavage rates was observed between Standard and Commercial treatment groups [68.1 (32/47) v. 79.2% (57/72), respectively; P = 0.25], nor in morula plus blastocyst rates on Day 7 (36.2 v. 45.8%, respectively; P = 0.39). However, the rate of transferable embryos (grade 1 and grade 2) on Day 7 was higher in the Commercial group (38.9 v. 12.8%; P &lt; 0.01). Of the COC in the Commercial group, a higher number of morula plus blastocyst were observed to be compact good COC (&gt;3 layers of cumulus cells) than compact regular COC (1-3 layers of cumulus cells) (66.7 v. 31.0% respectively; P &lt; 0.05), along with a higher number of transferable embryos on Day 7 (60.0 v. 23.8%, respectively; P &lt; 0.05). In conclusion, wood bison oocytes collected during the anovulatory season from non-superstimulated, non-synchronized bison and matured in vitro using portable incubators were competent to develop to the morula and blastocyst stages following IVF and culture. These results are important for future plans that require transporting oocytes from remote collection sites to the IVF laboratory, particularly with respect to the effectiveness of commercial maturation media which does not require CO2 supplementation. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

158 The Effect of Follicular Wave Phase at Time of Ovum Pick-Up on Bovine Oocyte Cytoplasmic Maturation and Developmental Competence

During oocyte collection, follicular wave phase is unknown, although differences in follicle environment may have dramatic effects on oocyte quality. This project was performed to determine whether oocyte collection during different phases of the follicular wave affects oocyte competence. Oocytes were collected via transvaginal ultrasound guided oocyte aspiration from 18 cows, at 4, 8, and 12 days following dominant follicle removal, representing follicle wave emergence, peak, and atresia, respectively (160, 314, and 273 oocytes, respectively). Once recovered, oocytes were graded and assigned to either being held as immature or matured in vitro for 24 h. Oocytes were then stained in Mitotracker deep red, fixed and stained with an anti-IP3R1 primary antibody and an Alexa Fluor 488-conjugated secondary antibody, before being stained with DAPI, to identify mitochondria, inositol triphosphate receptor 1 (IP3R1), and chromatin respectively. Mitochondria were analysed based on cytoplasmic distribution and classified as peripheral (immature), diffuse, central (mature), or sparse. Expression of IP3R1 was measured as corrected total cell fluorescence in Image J (National Institutes of Health, Bethesda, MD, USA). Staining patterns were analysed using ANOVA. A subset of the matured oocytes was stained with Fluo-3 to measure cytoplasmic calcium levels. These oocytes were then parthenogenetically activated before being imaged again to view changes in calcium levels, and presumptive embryos were cultured for 4 days. Fluo staining was measured as intensity levels (none, slight, moderate, high) and differences in development and stain levels were analysed using the Kruskal-Wallis test. Although mitochondria location was unaffected by collection day, it was significantly affected by maturation status (P = 0.0036). However, oocytes showed incomplete mitochondrial maturation, with mitochondria residing in the diffuse orientation in the majority of oocytes. Expression of IP3R1 appeared to be more sensitive to treatment. Expression significantly increased as meiosis proceeded (P = 0.0081) and there was a significant difference in expression between oocyte collection days (P = 0.0026). The interaction between collection day and maturation status also had a significant effect (P = 0.048), with mature oocytes showing an increase in IP3R1 expression, most notable in those collected on Day 4. Oocyte quality had a notable effect on the ability of oocytes to progress through meiosis (P = 0.054) and on mitochondrial location (P = 0.053), with AB oocytes showing better maturation parameters in both respects. Although the day of collection did not affect embryo development, Fluo stain intensity was an indicator of embryo developmental potential (P = 0.053), with oocytes having decreased potential to develop if the initial calcium levels were moderate to high. Results suggest that oocyte collection during wave emergence yields a slight advantage in oocyte quality. Although IP3R1, necessary for Ca2+ spikes during fertilization, indicates competence, high levels of cytoplasmic Ca2+ at the time of activation appear to be detrimental to embryo development.

ACEH CATTLE FOLLICLE DYNAMIC UNDER ENVIRONMENTAL HEAT STRESS

The objective of this research is to know Aceh cattle follicle dynamic under environmental heat stress condition. This research was conducted on December 2012 until August 2013. Animals used in this research were 20 Aceh cattle aged 5-8 years old, weighing of 150-250 kg, and had at least 2 regular cycles. All cattle used were clinically in good body condition score. The cattle were divided into two groups of 10, used in two different period of times (December-January and July-August) to examine the effect of environmental heat to their follicle dynamic. Each group is separated into two different keeping management; one group was kept in pens while the others in the pasture, 5 cattle in each management group. Research timing was based on information acquired from Indrapuri Agency for Meteorology, Climatology, and Geophysics (BMKG) which predicted that extreme weather would last from July to August while December to January would be relatively normal in Aceh region. All cattle’s estrous cycle were synchronized by 5 mg/mL of PGF2α intramuscularly (Lutalyse™, Pharmacia &amp; Upjohn Company, Pfizer Inc.). Ultrasonography examination was performed to monitor ovary follicle’s growth and dynamic during one cycle. Days during ovulation marked by standing heat was regarded as Day 0 of estrous cycle. Follicular dynamic examination during estrous cycle on December-January and also on July-August, both penned or pastured cattle showed the follicle waves that was 3 follicle growth waves. The size of follicle growth on first wave (1st DF) on cattle kept in pen on July-August and December-January were relatively similar. The size of follicle growth on first wave for cattle kept in pasture on July-August and December-January were relatively similar. To conclude, Aceh cattle’s follicle dynamic is not change during environment heat stress condition and is not affected by different cattle-keeping management.

Uterine endometrial vascularization during ovarian follicular growth in llamas: The effect of estradiol plasma concentration

The aim of this study was to evaluated changes in endometrial vascularization area (EVA) between the left and right uterine horn: a) during the ovarian follicular growth in intact llamas, and b) after exogenous estradiol administration of estradiol benzoate in ovariectomized (OVX) llamas. In experiment 1 follicle wave emergence was synchronized (n = 5 llamas) by follicle ablation (Day 0). Females were examined every other day from Day 1 to Day 27, using B mode ultrasonography to evaluate dominant follicle growth profile. Also, EVA was evaluated in each horn using Color-Power Doppler. Blood samples were taken every other day from Day 1 to Day 27 to measure estradiol (E2) plasma concentration by RIA. In experiment 2 OVX llamas (n = 4 llamas/group) were given a single im administration of: a) 1 mg of estradiol benzoate (EB) or b) 1 mL of saline. Females were subjected to ultrasound examinations every 48 h from Day −4 until treatment (Day 0), every 12 h from Day 0 to Day 4, and again every 48 h from Day 5 to Day 11. Evaluation of EVA in both uterine horns was performed as described for experiment 1. Blood sampling for the measurement of E2 was carried out at the same time points indicated for the ultrasound examinations. Serial data were analyzed by one way ANOVA for repeated measures using the MIXED Procedure in SAS. Also, Pearson's correlation was used to determine the relationship between variables. In intact llamas there was an effect of day on the dominant follicle growing profile (P < 0.01) and estradiol plasma concentration (P < 0.05). Dominant follicle diameter positively correlated (r = 0.4; P < 0.017) with estradiol plasma concentration. Also, EVA of right and left uterine horn did not differ (P = 0.89) during the evaluation period; however, it was affected by time (P < 0.05). In ovariectomized llamas estradiol concentration was significantly (P < 0.001) affected by treatment, time and their interaction. Accordingly, treatment with EB (P < 0.0001), time (P < 0.05) and their interaction (P < 0.01) affected EVA of both uterine horns; however, this variable did not differ between horns (P = 0.98). In conclusion, circulating concentrations of estradiol determined an increase in uterine vascularization, during the phase of follicular growth in intact llamas and after the exogenous administration of EB to ovariectomized females; however, no differential effect in endometrial vascularization area between right and left uterine horn was observed.

Aromatase inhibitors: A new approach for controlling ovarian function in cattle

Numerous treatments and protocols have been used to control the reproductive cycle in cattle, with varying effectiveness and many involving the administration of steroid hormones. Steroid hormones, such as estradiol, are perceived as having a negative impact on consumer health. This internationally shared opinion has led to a ban on the use of steroid hormones in food producing animals in many countries (i.e., European Union, New Zealand, and Australia). Letrozole, a non-steroidal aromatase inhibitor, inactivates the aromatase enzyme responsible for the synthesis of estrogens by reversibly binding to the “heme” group of the P450 subunit. Letrozole is approved as an adjuvant or first-line treatment for hormone-dependent breast cancer in post-menopausal women, but has been used increasingly for ovulation induction in the treatment of infertility in women. Using the bovine model to determine the effects on ovarian function, letrozole treatment was found to extend the lifespan of the dominant follicle and thereby delay emergence of the next follicle wave and/or ovulation. Letrozole treatment also had a luteotrophic effect; that is, larger CL and/or higher circulating concentrations of progesterone were detected in letrozole-treated heifers. Results of the initial studies in cattle provided the impetus for the development of aromatase inhibitor-based synchronization and fertility treatment in cattle. Biologically active concentrations of letrozole were achieved via intravenous, intramuscular or intravaginal administration, but the intravaginal route of administration is of particular interest because it permits extended and defined treatment periods, is minimally invasive, and reduces animal handling. Recent results revealed that irrespective of the stage of the cycle, a 4-day letrozole-based protocol induced ovulation in a significantly greater proportion of animals and with significantly greater synchrony than the control treatment. Evidence and reasons for the increasing use of programmed breeding and fixed-time artificial insemination are discussed in this review as a background to current development of an innovative aromatase inhibitor-based protocol as a safe and effective method of controlling the estrous cycle and ovulation in cattle.

Follicles and gonadotropins during waves 2 and 3 in three-wave interovulatory intervals in Bos taurus heifers

Observations were made on follicle dynamics and gonadotropin concentrations in anovulatory wave 2 and ovulatory wave 3 in three-wave interovulatory intervals (n = 15). Hypotheses were not used owing to inadequate availability of rationale. The future dominant follicles for waves 2 and 3 were designated DF2 and DF3 and the largest future subordinate follicles as SF2 and SF3, respectively. The day of expected diameter deviation (day 0) was defined as the day that DF2 or DF3 was closest to 8.5 mm. The first day that DF2 became smaller (P < 0.05) than DF3 was day 2 (10.7 ± 0.2 mm vs 11.8 ± 0.3 mm). The FSH surges 2 and 3 that stimulated waves 2 and 3 were similar at peak concentration, but the postsurge nadir of surge 2 occurred 1 day earlier than for surge 3. An LH increase was not temporally associated with deviation in wave 2, but an increase (P < 0.05) in LH in wave 3 began on day −1. Diameter of SF2 (6.5 ± 0.2 mm) on day 0 was less (P < 0.005) than for SF3 (7.2 ± 0.2 mm). Mean diameter of subordinate follicles in wave 2 did not differ among days. Diameter of subordinate follicles that attained ≥6 mm in wave 3 was greater (interaction, P < 0.02) by day 3 when in the right ovary (RO, 7.4 ± 0.2 mm) than when in the left ovary (LO, 5.6 ± 0.2 mm). The frequency of a conventional classification of deviation (future SF greater than 7.0 mm on day 0) was less (P < 0.001) for wave 2 (1 of 15 waves) than for wave 3 (8 of 15 waves). Novel observations involving DF2 and DF3 were (1) before deviation, diameter of DF2 vs DF3 and an incline in FSH surge 2 vs surge 3 were similar and (2) after deviation, smaller diameter of DF2 vs DF3 by day 2 was associated with an earlier cessation (nadir) in FSH surge 2 vs surge 3 and an absence of an LH increase during deviation. Novel observations involving subordinate follicles ≥6 mm were (1) before deviation, diameters were similar between waves 2 and 3 in association with the similar incline in FSH surges 2 vs 3 and (2) after deviation, a greater diameter increase of subordinates occurred in RO than in LO for wave 3, but an increase did not occur for either ovary in wave 2. The characteristics of diameter deviation were profoundly different between waves 2 and 3 owing to a smaller SF2 than SF3 at deviation but similar diameter of DF2 and DF3.

448 The effects of a novel follicle wave and heat synchronization protocol on the follicles, circulating hormones, and estrus of Aanestrous ewes

Reproductive performance in seasonally anestrous ewes is poor even after the use of current controlled breeding protocols. Anestrous ewes treated with a medroxyprogesterone acetate sponge, for 12 or 14 d, and estradiol-17β (E2) have synchronized follicular wave emergence. The objective of this study was to determine the effects of an E2 injection 6 d after CIDR insertion on follicles, E2 and LH concentrations, and estrus in seasonally anestrous ewes. Ewes (n = 13) received CIDRs (Day -12) followed by an injection of eCG (500 IU; i.m.; Day 0) at CIDR removal and an injection of sesame oil without (1 mL; i.m.; Control) or with E2 (350µg; i.m.; Day -6) 6 d before CIDR removal. Treatments were balanced for age, parity, and BCS. Blood samples were collected every 6 h for 48 h after E2 injection and every 3 h for 60 h starting 18 h after eCG injection to determine LH concentrations. Blood samples were collected on Day -6 and 0 and every 6 h for 60 h starting 18 h after eCG injection to determine E2 concentrations. Ovarian ultrasonography was done on Day -6 and 0. A ram was introduced on Day 1 to observe estrus. Ewes treated with E2 had higher LH concentrations (0.37 ± 0.05 ng/mL) than ewes treated with oil alone (0.12±0.05 ng/mL) during the 48 h after E2 injection (P < 0.005). Concentrations of LH were highest at 18 h after E2 injection (1.11 ± 0.12 ng/mL; P < 0.001). Ewes treated with E2 had higher LH concentrations (2.10 ± 0.18 ng/mL) than ewes treated with oil alone (0.12 ± 0.16 ng/mL) at 18 h after E2 injection (P < 0.001). The interval from eCG injection to the start of the pre-ovulatory LH surge was similar (37.1 ± 4.4 h; P > 0.05), but was more synchronized in ewes treated with E2 (2.3 ± 0.6 h) than ewes treated with oil alone (12.0 ± 5.1 h; P < 0.01). Concentrations of E2 were similar between treatments on Day -6 (2.1 ± 0.3 pg/mL; P > 0.05) and Day 0 (2.3 ± 0.4 pg/mL; P > 0.05). Concentrations of E2 tended to be highest at 18 h after eCG injection (3.5 ± 0.5 pg/mL; P = 0.076). Maximum follicular diameter was larger on Day 0 (4.8 ± 0.2 mm) than Day -6 (3.4 ± 0.2 mm; P < 0.001). There were no differences in the interval from eCG treatment to the start (37.6 ± 2.7 h) or end (74.7 ± 1.9 h) of estrus (P > 0.05). Adding an E2 treatment during a CIDR-eCG estrus synchronization protocol increased LH concentrations after E2 treatment and stimulated a more synchronized pre-ovulatory LH surge in seasonally anestrous ewes.

Follicular waves and hormonal profiles during the estrous cycle of carriers and non-carriers of the Trio allele, a major bovine gene for high ovulation and fecundity

A high fecundity bovine genotype has recently been discovered and genetic mapping indicates evidence for segregation of a major gene with influence on ovulation rate located on bovine chromosome 10. Cattle carrying the high fecundity allele, referred to as the Trio allele, have multiple ovulations while half-sibling, non-carriers generally have single ovulations. The present study was designed to evaluate follicle wave patterns and associated circulating hormones during the estrous cycle of Trio allele carriers (n = 7) and non-carrier half-sib controls (n = 5). We hypothesized that Trio allele carriers would exhibit multiple smaller dominant follicles and greater circulating FSH than non-carrier controls. The proportion of Trio carrier and non-carrier cows with 2 or 3-wave patterns was not different between genotypes with the majority (>70%) exhibiting 3-wave patterns. Trio carriers had greater (P < 0.01) number of ovulations (∼4 vs ∼1 ovulations) and smaller preovulatory follicles (8.9 vs. 14.9 mm; P < 0.01) than non-carrier controls. However, total luteal tissue volume and circulating progesterone, normalized to the initial ovulation or to the onset of luteolysis, were not different between genotypes (P > 0.10). Follicular waves were found to be associated with an FSH surge in both genotypes. Peak FSH concentration at each follicular wave (3-wave patterns) was not different (P > 0.05) between genotypes, but circulating FSH during the decline and nadir, encompassing the day of deviation, was greater (P < 0.05) in Trio carriers. Despite a difference (P = 0.032) in the length of the estrous cycle (23.0 vs. 25.2 d; Trio carrier and non-carriers respectively), the pattern of follicle growth, such as day of wave emergence, day of follicle deviation, and day of maximum diameter of the dominant follicle, when normalized to the FSH surge of each follicular wave were similar in Trio carriers compared to non-carriers although Trio carriers consistently had much smaller-sized follicles (P < 0.05). Thus, decreased follicle size and greater circulating FSH are key components of the mechanism that produces multiple ovulations in cattle that are carriers of the Trio high fecundity allele.

Screening candidate microRNAs (miRNAs) in different lambskin hair follicles in Hu sheep.

Hu sheep lambskin is a unique white lambskin from China that exhibits three types of flower patterns, including small waves, medium waves, and large waves, with small waves considered the best quality. However, our understanding of the molecular mechanism underlying flower pattern formation in Hu sheep lambskin is limited. The aim of the present study was to further explore the relevance between candidate microRNAs (miRNAs) and developmental characteristics of hair follicles and screen miRNAs for later functional validation. Herein, we employed Illumina Hiseq 2500 to identify differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves to construct a comprehensive sequence database on the mechanism of hair follicle development. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of candidate miRNAs in different flower patterns were analyzed by relative quantitation using real-time PCR, combined with histological observation and micro-observation technologies, and the correlation between expression levels of candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0. A total of 522 differentially expressed miRNAs were identified, and RNA-seq analysis detected 7,266 target genes in different groups of flower patterns. Gene ontological analysis indicated these target genes were mainly involved in cell proliferation, differentiation, growth, apoptosis, and ion transport, and 14 miRNAs, including miR-143, miR-10a, and let-7 were screened as candidate miRNAs in Hu sheep hair follicle growth and development. In the same field of vision, variance analysis showed that the number of secondary follicles in small waves was significantly larger than that in large and medium waves (P<0.01); the diameter of the primary and secondary follicles in large waves was respectively larger than those in medium and small waves (P<0.01). Combined with correlation analysis between miRNA expression and histological properties of hair follicles, highly significant differences in miRNA-143 expression levels between large and small waves were observed (P<0.01), and significant differences in the miRNA-10a expression levels between large and small waves (P<0.05) and in let-7i expression levels between large and medium waves were observed (P<0.05). Significant differences in the expression of novel miRNAs of NW_004080184.1_6326 between medium and large waves were detected (P<0.05), and highly significant differences between medium and small waves were observed (P<0.01). Highly significant differences in the expression level of NW_004080165.1_8572 between medium and large and small waves (P<0.01), in that of NW_004080181.1_3961 between medium and small waves (P<0.01), and in that of NW_004080190.1_13733 between medium and large waves were observed, whereas no significant differences in the other miRNAs among large, medium, and small waves were detected. Overall, the present study showed that miRNA-143, miRNA-10a, let-7i, NW_004080184.1_6326, NW_004080165.1_8572, NW_004080181.1_3961, and NW_004080190.1_13733 could be considered as important candidate genes, indicating these seven miRNAs may play significant roles in hair follicle growth and development in Hu sheep lambskin.

Inducing sub-luteal circulating concentrations of progesterone and persistent follicles in Holstein heifers and non-lactating cows using a modified CIDR device

Reducing progesterone from Controlled Internal Drug Release (CIDR) devices may allow for the comparison of contrasting steady state concentrations of progesterone (P4) in non-lactating dairy cows and heifers. This technical study was designed to determine if circulating P4 concentrations from the CIDR device could be reduced in cattle via ethanol extraction, and if reduced steady state concentrations of P4 would increase the percent of cows and heifers that developed a persistent follicle. Holstein heifers and non-lactating cows were treated with Ovsynch (100μg GnRH, 7 d later, 25mg dinoprost tromethamine, 2 d later, 100μg GnRH) to initiate a new cycle. Only heifers and cows that synchronized were included in this study. On d 7 of the newly induced estrous cycle, animals were assigned to three treatments all treated with new CIDRs as follows: CIDR-N, CIDR that was not manipulated; CIDR-W, CIDR immersed in water, or CIDR-E, CIDR immersed in a mixture of equal parts water/ethanol. All heifers and cows received 25mg dinoprost tromethamine at time of treatment to ensure regression of the d 7 corpora lutea (CL). Only data from heifers and cows having morphological CL regression observed with ultrasonography on d 1 and 2 following treatment, and with basal P4 concentrations (<0.15ng/mL) on d after CIDR removal, were considered in the final analyses (n =30). CIDR-E had lower circulating concentrations of P4 during the treatment period compared to CIDR-N and CIDR-W in cycling heifers and non-lactating cows and a significant interaction of treatment x d from CIDR insertion was observed. CIDR-W had lower P4 compared to CIDR-N in heifers, but not in cows. With the three treatments combined, non-lactating cows had lower circulating concentrations of P4 than heifers. The 1st wave dominant follicle became atretic followed by a new wave of follicles in 4 of 5 non-lactating cows and heifers treated with CIDR-N compared to 1 of 12 in the CIDR-E and 3 of 13 in CIDR-W treatments during the 7 d treatment period. In summary, reduced steady state serum concentrations of progesterone from a CIDR device resulted in continued growth of persistent follicles in cows and heifers compared to a non-manipulated CIDR device.

Effects of a single 20\xa0mg dose of letrozole on ovarian function post dominant follicle selection: an exploratory randomized controlled trial

BackgroundOur objective was to explore the impact of a single dose of an aromatase inhibitor (letrozole) administered at defined times of the follicular phase or immediately after ovulation on dominant follicle development, luteogenesis and new follicle wave emergence.MethodsA prospective pilot study using a randomized complete block, controlled, open label design was conducted at an academic clinical research center. Forty-five healthy, female volunteers (25.5 ± 0.9 years, BMI 25.0 ± 0.6 kg/m2) who had not taken hormonal contraceptives for a minimum of 2 months were recruited. A 20 mg dose of Letrozole was administered once orally in each of 3 groups when the dominant follicle reached a diameter of 1) 12 mm, 2) 18 mm, 3) the first day following ovulation (post-ovulation), or 4) treatment was withheld (control). Serial ultrasonography and phlebotomy began on day 4 of the menstrual cycle and continued for 1.5 menstrual cycles. Participants recorded menses and daily events in a life events calendar for the duration of the study. Demographic and single point measurements were compared among groups by ANOVA. Changes in hormone concentrations over time were compared among groups by repeated measures ANOVA. Kruskal-Wallis tests were used for non-normally distributed data.ResultsThe dominant follicle in all treatment groups ovulated. There were no differences among experimental groups in peak follicle diameter, follicular growth rate, endometrial thickness at ovulation or inter-ovulatory interval. Plasma concentrations of estradiol dropped, while FSH and LH concentrations rose following treatment in all treatment groups. Plasma FSH and LH concentrations were higher in the 18 mm group compared to the 12 mm and post-ovulation groups (P < 0.02).ConclusionAdministration of a single 20 mg dose of Letrozole at the times of the menstrual cycle we examined did not induce dominant follicle regression or failure of corpus luteum formation. Letrozole-induced suppression of estradiol synthesis by the dominant follicle was not detrimental to follicle growth or ovulation following follicle selection, likely due to increased circulating concentrations of FSH and LH resulting from a lack of estradiol-induced suppression of the hypothalamic-pituitary-ovarian axis.Trials registration numberClinical trials registration number NCT01046578.

Incidence of double ovulation during the early postpartum period in lactating dairy cows

In lactating cattle, the incidence of twin calving has many negative impacts on production and reproduction in dairy farming. In almost all cases, natural twinning in dairy cattle is the result of double ovulation. It has been suggested that the milk production level of cows influences the number of ovulatory follicles. The objective of the present study was to investigate the incidence of double ovulations during the early postpartum period in relation to the productive and reproductive performance of dairy cows. The ovaries of 43 Holstein cows (26 primiparous and 17 multiparous) were ultrasonographically scanned throughout the three postpartum ovulation sequences. The incidence of double ovulation in the unilateral ovaries was 66.7%, with a higher incidence in the right ovary than in the left, whereas that in bilateral ovaries was 33.3%. When double ovulations were counted dividing into each side ovary in which ovulations occurred, the total frequency of ovulations deviated from a 1:1 ratio (60.3% in the right side and 39.7% in the left side, P < 0.05). In multiparous cows, double ovulation occurred more frequently than in primiparous cows (58.8% vs. 11.5% per cow and 30.0% vs. 3.8% per ovulation, respectively P < 0.01). The double ovulators experienced more anovulatory repeated waves of follicles before their first ovulations than the single ovulators, which resulted in an extension of the period from parturition to third ovulation (81.5 days vs. 64.2 days, P < 0.05). In the multiparous cows, the double ovulators exhibited higher peak milk yield (P < 0.01) with lower milk lactose concentration (P < 0.05), indicating the prevalence of a more severe negative energy balance during the postpartum 3-month compared to the multiparous single ovulators. Our results showed that, regardless of their parity, double ovulation had no impact on the reproductive performance of the cows. Two multiparous cows that experienced double ovulation during the early postpartum period subsequently conceived twin fetuses. It can be speculated that the incidence of double ovulations during the early postpartum period partly contributes to the increased incidence of undesirable twin births in multiparous dairy cows.

In vivo and in vitro maturation of oocytes collected from superstimulated wood bison (Bison bison athabascae) during the anovulatory and ovulatory seasons

Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day −1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days −9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60–70% of oocytes were at the MII stage 24h after in vitro maturation while only 25–27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.

Genomic portrait of ovarian follicle growth regulation in cattle

The female reproductive cycle is characterized by cyclic patterns of growth of ovarian follicles destined to ovulate and the degeneration of those not chosen for ovulation. Apoptosis is the underlying mechanism of ovarian follicle degeneration during atresia. The fate of ovarian follicles is under the control of endocrine hormones (e.g. gonadotropins) and intra-ovarian regulators (e.g. growth factors and gonadal steroids), but the intra-cellular and molecular regulation of follicle fate (cellular proliferation or atresia) are still not fully understood. Studies using genomic tools which aim to determine the expression profiles of mRNAs and miRNAs in bovine dominant and subordinate ovarian follicles at different stages of the follicle wave have indicated that there are some novel molecular factors (e.g. FOXO1 and miR-183∼96∼182 cluster) associated with ovarian follicle development. Furthermore, bioinformatics tools have been used to identify the interactions between mRNAs and miRNAs leading to better understanding of ovarian follicle growth regulation in cattle. This review summarizes the recent findings about the complex intracellular communication networks involving miRNA, their target genes and signalling molecules which together may coordinate the fate of cattle ovarian follicles.