Small Preovulatory Follicles Research Articles

Ovarian response and conception rate in Boran and Boran*Holstein cows treated by Gonadotrophin-realizing hormone and ProstaglandinF2α with and without exogenous progesterone

BackgroundDifference in breed, nutrition status and climate in which animals are managed result in differences in response to reproductive hormones. Fertility rate to artificial insemination is very low in Ethiopian Boran and Boran*Holstein crosses. This partly maybe due to adopting estrus and/or ovulation synchronization developed for temperate taurine cattle. Experimental study was conducted to evaluate ovarian response to combinations of Gonadotrophin-Realizing Hormone agonist (gonadorelin) and ProstaglandinF2α (PGF2α) with or without progesterone (Controlled Internal Drug Release/CIDR), and conception rate to timed AI. Postpartum native Ethiopian Boran (n = 60) and Boran*Holstein cross (n = 66) cows were randomly assigned to four treatment groups as Ovsynch (gonadorelin on day of start, PGF2α seven days later, 2nd gonadorelin at 48 h of PGF2α and insemination at 19 h of the 2nd gonadorelin); CIDR + Ovsynch (same as Ovsynch but CIDR device was inserted into vagina for 7 days); Cosynch (same as Ovsynch but insemination was made at the 2nd gonadorelin) and CIDR + Cosynch (same as Cosynch but CIDR was inserted for 7 days).ResultThere was no difference (P > 0.05) in ovulation rate to day 9 gonadorelin (88.33% in Boran; 78.79% in Boran*Holstein) and interval from day 9 gonadorelin to ovulation (36.5 ± 1.13 h in Boran and 36.057 ± 1.11 h in Boran*Holstein). Dominant follicle immediate to ovulation (14.95 ± 0.19 mm Vs 19.12 ± 0.49 mm) and corpus luteum size (16.31 ± 0.33 mm Vs 20.28 ± 0.43 mm ) respectively were smaller (P < 0.05) in Boran than Boran*Holstein. Plasma progesterone concentration at PGF2α was higher (P < 0.05) in Boran (11.91 ± 0.74ng/mL) than Boran*Holstein (6.13 ± 0.27ng/mL) but luteolysis rate was lower (P < 0.05) in Boran (87.9%) than Boran-Holstein (96.9%). Cows with CIDR had higher conception rate than cows without CIDR (72.00% Vs 39.02% in Boran*Holstein and 74.07%, Vs 51.52% in Boran respectively). Insemination at 19 h of gonadorelin administration resulted in higher conception rate (78.6% for Boran; 71.43% for Boran*Holstein) than insemination at gonadorelin (69.29% for Boran; 66.67% for Boran*Holstein).ConclusionBoran cows have smaller preovulatory follicles, smaller corpus luteum, large amount of progesterone and lower rate of luteolysis to PGF2α compared to Boran*Holstein. The CL of Boran cattle seems les reactive to PGF2α than Boran*Holstein CL. CIDR significantly improved conception rate in Boran and Boran*Holstein cows.

Postponing TAI in beef cows with small preovulatory follicles

The aims of this study were (1) to establish an optimal cut-off point for evaluating the effect of the preovulatory follicle (POF) diameter in timed AI (TAI) programs, and (2) to evaluate the effect of postponing TAI in cows with smaller follicles on pregnancy per AI (P/AI). In Study 1, Nelore cows (n = 426) were subjected to an estradiol-progesterone (P4) based TAI protocol. The diameter of POF was measured at TAI, 48 h after P4 insert removal. From the ROC curves for determining the relationship between the POF diameter and pregnancy, the cut-off was 11 mm (P < 0.001). Cows with larger POF had greater (P < 0.05) P/AI (62.5%) than cows with smaller POF (34.8%). In Study 2, Nelore cows (n = 1257) were subjected to the same TAI protocol as in Study 1. Before TAI, cows were separated according to POF diameter in three groups: (1) Larger POF (LP, n = 873; POF ≥11 mm), (2) Smaller POF 0 h (SP0, n = 195; POF <11 mm), and (3) Smaller POF 24 h (SP24, n = 189; POF <11 mm). Cows in the LP and SP0 were TAI at 48 h and cows in the SP24 group were TAI at 72 h after P4 insert removal. Cows in the LP group had the greatest (P < 0.05) P/AI (60.25%), followed by cows in the SP24 group (41.8%), while cows in the SP0 group had the lowest P/AI (31.8%). In conclusion, cows with smaller follicles have lower P/AI, but postponing the TAI by 24 h in these cows increases P/AI.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows.

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography—high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows.

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7–14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway “oxidative phosphorylation” was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

The Impact of Endocrine-Disrupting Chemicals on Female Fertility

Nowadays, infertility is a prevalent problem among couples. Exposure to endocrine-disrupting chemicals (EDCs) may influence the female reproductive outcomes through multiple mechanisms, being one of the important causes of infertility. These compounds may interfere with some fertility and fecundity markers, leading to increased time to pregnancy, increased risk of spontaneous abortion, shorter menstrual cycles, early age at menopause, anovulation or delayed ovulation, smaller preovulatory follicles, increased serum FSH, decreased synthesis of estradiol and progesterone, low plasma levels of anti-Mullerian hormone and decreased antral follicle count. Endocrine-disrupting chemicals may also impair in vitro fertilization outcomes, being responsible for: a lower probability of implantation, reduced fertilization rates, diminished probability of clinical pregnancy, and near term pregnancies after in vitro fertilization. Another mechanism of altering the female reproductive function is through the disorders induced by EDCs, such as polycystic ovary syndrome, uterine fibroids, and endometriosis. Those conditions may finally lead to infertility.

The follicular microenvironment in low (++) and high (I+B+) ovulation rate ewes.

Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.

Effect of sperm kinematics and size of follicle at ovulation on pregnancy rate after timed AI of beef cows

The objectives of this study were to evaluate whether the diameter of the preovulatory follicle and the post-thaw sperm kinematics are factors that affect successful establishment of a pregnancy after timed AI (TAI) of postpartum beef cows. Nelore cows (n = 346) were subjected to an estradiol-progesterone based TAI protocol. At TAI, cows were categorized according to the diameter of the preovulatory follicle (POF) in the Early Ovulation (EO group, n = 184), and Late Ovulation (LO group, n = 162) groups. Sperm were classified, according to CASA analysis, as Hyper-activated (H+), and Non-Hyper-activated (H-). The H + and H- semen was used to inseminate cows that were classified to be in the EO and LO groups. Thus, after distribution, the groups were: EOH+ (n = 89), EOH- (n = 95), LOH+ (n = 96), and LOH- (n = 66). Pregnancy per AI (P/AI) was greater (P < 0.05) for EOH+ (66.3%, 59/89), EOH- (65.3%, 62/95), and LOH- (72.7%, 48/66) groups than for LOH + group (52.1%, 50/96). The results of the study indicate that cows with a smaller POF are less likely to become pregnant when insemination is with semen considered as hyper-activated (H+). In contrast, when inseminations are performed with semen categorized to be non-hyper-activated (H-), cows of the EO and LO groups had acceptable and similar pregnancy rates as a result of timed AI.

Effect of preovulatory follicle on fertility in Graded murrah buffaloes (Bubalus bubalis)

An investigation was taken up to study the relationship of preovulatory follicle size at the time of first postpartum AI and CL biometry with conception using transrectal ultrasonography in 70 parous Graded Murrah buffaloes maintained under rural conditions. POF sizes were measured ultrasonographically and grouped in to small preovulatory follicle (SPF), medium preovulatory follicle (MPF) and large preovulatory follicle (LPF). Serum progesterone concentration at the time of AI and pregnancy status was negatively correlated indicating that when progesterone level declined to less than 0.3 ng/ml (basal level) at the time of AI, the chances of the animal becoming pregnant was increased. The size of the POF was positively correlated to the size of the CL on day 10 in pregnant buffaloes. The overall POF diameter at the time of estrum was 12.31 ± 0.29 mm (Range 9 to 16 mm) in Graded Murrah buffaloes and 50 % of the buffaloes had POF size &gt; 12 - £14 mm. They conceived only if the POF diameter was more than 9 mm at the time of AI however there was no significant correlation between the POF size at the time of AI and conception. Thus, it was concluded that physiological maturity rather than the diameter of the follicle influenced the fertility in Graded Murrah buffaloes under field conditions.

Evidence of endometrial amino acid metabolism and transport modulation by peri-ovulatory endocrine profiles driving uterine receptivity

BackgroundIn beef cattle, changes in the periovulatory endocrine milieu are associated with fertility and conceptus growth. A large preovulatory follicle (POF) and the resulting elevated concentrations of progesterone (P4) during diestrus positively affect pregnancy rates. Amino acids (AA) are important components of maternally derived secretions that are crucial for embryonic survival before implantation. The hypothesis is that the size of the POF and the concentration of P4 in early diestrus modulate the endometrial abundance of SLC transcripts related to AA transport and metabolism and subsequently impact luminal concentrations of AA. The follicle growth of Nelore cows was manipulated to produce two experimental groups: large POF and CL (LF-LCL group) and small POF and CL (SF-SCL group). On Day 4 (D4; Experiment 1) and Day 7 (D7; Experiment 2) after GnRH-induced ovulation (GnRH treatment = D0), the animals were slaughtered and uterine tissues and uterine washings were collected. qRT-PCR was used to evaluate the expression levels of AA transporters in D4 and D7 endometrial tissues. The concentrations of AA were quantified in D4 and D7 uterine washings by HPLC.ResultsTranscript results show that, on D4, SLC6A6, SLC7A4, SLC17A5, SLC38A1, SLC38A7 and SCLY and on D7 SLC1A4, SLC6A1, SLC6A14, SLC7A4, SLC7A7, SLC7A8, SLC17A5, SLC38A1, SLC38A7, SLC43A2 and DDO were more abundant in the endometria of cows from the LF-LCL group (P < 0.05). In addition, concentrations of AA in the uterine lumen were influenced by the endocrine profiles of the mother. In this context, D4 uterine washings revealed that greater concentrations of taurine, alanine and α-aminobutyric acid were present in SF-SCL (P < 0.05). In contrast, lower concentrations of valine and cystathionine were quantified on D7 uterine washings from SF-SCL cows (P < 0.05).ConclusionThe present study revealed an association between the abundance of transcripts related to AA transport and metabolism in the endometrium and specific periovulatory endocrine profiles related to the receptive status of the mother. Such insights suggest that AAs are involved in uterine function to support embryo development.

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.

Relationship of Body Condition Score at Estrus and Conception Rate in Graded Murrah Buffaloes

Seventy graded Murrah parous buffaloes presented for the first postpartum AI maintained under village system of rearing free from apparent pathological abnormalities of the reproductive tract were selected to study the effect of body condition score (BCS) and size of the preovulatory follicle on conception rate. After evaluating Body condition score (BCS) by visual examination and estrus intensity based on the score card, Preovulatory follicle (POF) sizes were measured ultrasonographically and grouped as small preovulatory follicle (SPOF), medium preovulatory follicle (MPOF) and large preovulatory follicle (LPOF). The mean values of BCS, intensity of estrus and serum progesterone at AI and on day 10 post AI were estimated and the relation to conception rate was analyzed. Positive correlation was observed with the body condition of the buffaloes at the time of AI to POF size and intensity of estrus. The mean values of BCS in pregnant and non pregnant buffaloes did not differ significantly and it has a nonsignificant (P>0.05) negative correlation with pregnancy status, however proper nutrition during breeding season is necessary for acceptable reproduction. It was concluded from the present study that physiological maturity of the follicle rather than its diameter influenced the fertility in graded Murrah buffaloes under field conditions.

Timed artificial insemination in blocks: A new alternative to improve fertility in lactating beef cows

The aim of this study was to evaluate whether changing the interval from CIDR removal to timed artificial insemination (TAI) according to the diameter of the preovulatory follicle (POF) would improve pregnancy per AI in cows. In Study 1, a retrospective analysis of TAI experiments (n=96 cows) was performed to characterize the time of ovulation according to the diameter of the dominant follicle. It was observed that cows with a larger POF had ovulations earlier than cows with smaller POF, according to the equation: y=0.72x2−26.74x+264.54 (R2=0.63; P<0.001). In Study 2, lactating Nelore cows (n=412) were subjected to an EB-CIDR based TAI protocol. On the morning of Day 10 (time of TAI), cows were randomized into Control (n=209) and Block (n=203) groups; (1) Cows in the Control Group were TAI 48h after CIDR removal (08:00am on Day 10), and; (2) Cows in the block group were inseminated once at one of the following time points, according to the diameter of the POF on Day 10: B0 (POF≥15mm, TAI 0h after convetional TAI), B1 (POF 13–14.9mm, TAI 6h later), B2 (POF 10.1–12.9mm, TAI 24h later) and B3 (POF≤10mm, TAI 30h later). The cows of the Block Group had greater pregnancy rates per AI than the Control Group (129/203, 63.5% when compared with 102/209, 48.8%, respectively; P<0.01). In conclusion, results of the present study demonstrate that adjusting the timing of TAI according to the diameter of the POF can be an effective practice for improving fertility of cows in TAI protocols.

11 FERTILITY RESPONSE IN SUCKLED BEEF COWS SUPPLEMENTED WITH LONG-ACTING PROGESTERONE AFTER TIMED ARTIFICIAL INSEMINATION

Corpus luteum (CL) and progesterone (P4) secretion are affected by preovulatory follicle (POF) size. Increased circulating P4 during early diestrus has a positive effect on embryo development in beef cattle. However, the combined effects of the POF size and P4 supplementation during early diestrus on fertility of beef cows are not known. The objective was to evaluate the effects of POF size and supplementation of long-acting P4 after timed-AI on pregnancy rates (P/AI). Suckled Nelore cows (n = 596) were evaluated twice by transrectal Doppler ultrasonography (US) 10 days apart to detect the cyclic status. In Study 1, anestrous cows (absence of CL on both exams) received an intravaginal P4 device and an oestradiol benzoate (EB) injection on Day –10 (day of the second US). Devices were removed and sodium cloprostenol [prostaglandin F2α (PGF2α)], oestradiol cypionate, and eCG were given on Day –2. Cows were timed-AI on Day 0 and assigned to receive placebo (control group, n = 187) or 150 mg of long-acting P4 on Day 4 (P4 group, n = 189). In Study 2, cyclic cows (presence of CL) received a PGF2α injection on Day –20 (first US). Cows with a new CL on Day –10 received an intravaginal P4 device and an injection of EB and were split to receive an injection of PGF2α [large follicle (LF); n = 109] or not [small follicle (SF); n = 111]. Devices were removed and PGF2α was injected on Day –2. Ovulation was induced with buserelin acetate, and cows were timed-AI on Day 0 and split to receive placebo (LF/control group, n = 55, and SF/control group, n = 55) or 150 mg of long-acting P4 on Day 4 (LF/P4 group, n = 56, and SF/P4 group, n = 54). Ultrasonographic scanning was done on Days 0, 4, and between 35 and 40 to detect the POF and CL sizes and P/AI, respectively. Data were analysed using PROC GLIMMIX (SAS Institute Inc., Cary, NC). In anestrous cows, P/AI was reduced in POF with &lt;11 mm. The P/AI was greater in the P4-treated group than in the control group for all cows (55.6% v. 46.0%; P = 0.05) and for ovulated cows (59%, 105/178 v. 49%, 86/173; P = 0.08). For cyclic cows, POF size (mm) on Day 0 (13.5 ± 0.3 v. 11.2 ± 0.2), ovulation rate (90% v. 77%), and CL area (cm2) on Day 4 (1.46 ± 0.05 v. 1.25 ± 0.05) were greater (P &lt; 0.007) in the LF group than in the SF group. There was a main effect of follicle group on P/AI (54%, LF group v. 38%, SF group; P &lt; 0.01). Moreover, P/AI were greater (P &lt; 0.05) in the LF/control (56%) and LF/P4 (52%) groups than in the SF/control group (31%), whereas no difference was detected between the SF/P4 group (45%) and the other groups. Among cows that ovulated, P/AI was lower (P = 0.05) in the SF/control group (41%, 17/41) compared with the LF/control group (62%, 31/50) and were similar for the SF/P4 group (56%, 25/45) and LF/P4 group (57%, 28/49) compared with others. We suggest that P4-stimulated embryotrophic effects improved fertility in anestrous beef cows supplemented with long-acting P4 on Day 4 after timed-AI. Also, the presence of a functional CL during follicle growth results in smaller POF and CL and reduces the ovulatory and P/AI rates in cyclic cows. Post-AI P4 supplementation may attenuate the negative effects of small POF/CL. Research was supported by CNPq, FAPESP, Ouro Fino Agronegócio, and Innovare.

106 THE RECEPTIVE BEEF COW ENDOMETRIUM: POTENTIAL KEY FEATURES FAVOURING THE COMMUNICATION BETWEEN EMBRYONIC AND MATERNAL TISSUES

A receptive state of the endometrium is crucial for proper communication with the pre-implantation embryo and, thereby, a perquisite for a successful pregnancy. Both reduction of transmembrane mucin and initiation of apoptotic events have been proposed as key features prompting embryo/endometrial cell interaction and facilitating maternal receptivity of the murine and human embryo. In beef cows, however, the signature for receptivity needs to be elucidated. Therefore, we aimed to characterise “receptive” versus “refractory” endometrial tissues with the focus on (1) transcription profiles related to mucin, apoptosis and proliferative pathways, and 2) phenotypic features; that is, epithelial transmembrane mucin and apoptotic cell rates (ACR). Using a bovine model, preovulatory follicle growth was manipulated to produce 2 groups: cows with larger preovulatory follicle, longer proestrus, and higher receptivity (LF-LCL) versus cows with smaller preovulatory follicle, shorter proestrus, and lower receptivity (SF-SCL). Seven days post-induced ovulation, endometrium was collected. Transcriptome profiles of endometrial tissue (n = 3/group) were determined using Illumina RNA sequencing analyses. In addition, paraffin embedded endometrial samples (n = 6/group) were stained with Alcian Blue for semiquantitative analyses of mucin staining intensity and treated with antibodies against activated Casp3 to determine ACR. RNA-seq data showed that cell surface MUC1 gene expression was drastically reduced (fold-change –3.33) in LF-LCL tissue (P &lt; 0.05). In contrast, the LF-LCL endometrial tissue displayed an up-regulated expression of genes involved in apoptosis pathways, such as Casp9, CRADD, DAPL1 (fold-change 1.80, 2.10, and 3.60, respectively; P &lt; 0.05). Moreover, the expression of genes related to proliferation, such as MYC and NOV, was significantly down-regulated in LF-LCL endometrial tissue (fold-change –2.2 and –6.3, respectively; P &lt; 0.01). Histology of endometrial samples revealed that the signal for transmembrane, anti-adhesive, mucin at the LF-LCL epithelium was consistently low, whereas the SF-SCL tissue group displayed variable amounts of transmembrane mucin at the apical epithelial boarder. Immunohistochemistry showed that gland cells from LF-LCL endometrial tissue displayed a significantly higher ACR compared with gland cells of the SF-SCL tissue (29.6 ± 1.52% v. 11.9 ± 1.26%; P &lt; 0.05). In the surface epithelium and the stromal tissue, Casp3 positive cells were rare and ACR was similar between groups. In conclusion, a down-regulated expression of the transmembrane mucin might indicate a receptive condition of the endometrium. Furthermore, the prominent apoptotic characteristics in the LF-LCL tissue suggest that the receptive endometrium must surpass the proliferative status in order to differentiate to more specialised functions, such as embryotrophy.

9 TIMED ARTIFICIAL INSEMINATION IN BLOCKS: A NEW ALTERNATIVE TO IMPROVE FERTILITY IN BEEF COWS

The objective of this study was to evaluate whether timed artificial insemination (TAI) according to the diameter of the dominant preovulatory follicle (POF) would improve pregnancy rates in beef cows. In Experiment 1, a retrospective meta-analysis of 5 oestradiol- or gonadotropin-releasing hormone-based TAI experiments from 2011 to 2014 was performed to evaluate the interval from luteolysis to ovulation. In these experiments, crossbred cows (Gyr × Holstein; n = 60) were examined by ultrasonography at 12-h intervals from progesterone-releasing device (CIDR) removal to ovulation. A linear regression model was used to predict the effect of POF diameter on the time of ovulation. Cows with a larger POF at the time of AI ovulated earlier than cows with smaller POF (y = –8.25x + 115.22; R2 = 0.93; P &lt; 0.0001). In Experiment 2, lactating Nelore cows (Bos indicus; n = 59) on random days of the oestrous cycle were given 2 mg of oestradiol benzoate IM and a CIDR device (Day 0) to synchronize follicular waves. The CIDR were removed, and cows were given 500 μg of d-Cloprostenol (prostaglandin F2α) IM, 1 mg of oestradiol cypionate IM, and 300 IU of eCG IM on Day 8. On the morning of Day 10 (07:00 a.m.), the diameter of the POF was assessed by ultrasonography, and cows were randomised into Control (n = 29) and Block (n = 30) groups. 1) Control-group cows were TAI 48 h after CIDR removal (08:00 a.m. on Day 10), and Block-group cows were inseminated at 4 time points according to the diameter of the POF: B0 (POF ≥15 mm, TAI at 08:00 a.m. on Day 10, n = 6), B1 (POF 13 to 14 mm, TAI at 02:00 p.m. on Day 10, n = 8), B2 (POF 11 to 12 mm, TAI at 08:00 a.m. on Day 11, n = 11), and B3 (POF ≤ 10 mm, TAI at 02:00 p.m. on Day 11, n = 5). Pregnancy status was assessed 30 days post-AI by ultrasonography. No differences were detected in the diameter of the POF 48 h after CIDR removal (P = 0.77) or ovulation rate (P = 0.65; combined ovulation rate 52/59, 88%) between Control and Block groups. Block group had a higher pregnancy rate than Control (22/30, 73% v. 13/29, 45%, respectively; P = 0.02). In conclusion, our results document that interval from luteolysis to ovulation depends on the size of preovulatory follicle, timing of AI in relation to ovulation time is critical, and AI time according to the diameter of the POF can be an effective tool to improve fertility of cows in TAI protocols.

Modulation of periovulatory endocrine profiles in beef cows: consequences for endometrial glucose transporters and uterine fluid glucose levels

In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus.

Successful pregnancy in a woman with Kallmann's syndrome using human menopausal gonadotropin followed by low-dose human chorionic gonadotropin in the mid-to-late follicular phase

Kallmann’s syndrome is characterized by hypogonadotropic hypogonadism and anosmia [1]. It is a rare disorder, occurring in only one per 50,000 women [2]. Induction of ovulation in women with hypogonadotropic hypogonadism requires follicle-stimulating hormone (FSH) for follicular growth, and both FSH and luteinizing hormone (LH) to induce optimal follicular steroidogenesis and ovulation. Numerous reports of achieving successful pregnancies in women with Kallmann’s syndrome have appeared in the literature. Recent evidence suggests that low-dose human chorionic gonadotropin (hCG) can also be used to mimic LH actions on developing follicles in a more sustained and stable manner, permitting the progression of folliculogenesis when the LH/ hCG receptors begin to be expressed in the granulose cells of larger ovarian follicles [3]. More specifically, serum levels of hCG during human menopausal gonadotropin (hMG) administration were inversely correlated with the occurrence of small preovulatory follicles [4]. We describe here a woman with Kallmann’s syndrome who was treated with a combination of hMG and low-dose hCG to achieve ovulation induction and successful pregnancy. A 29-year-old woman was referred to our hospital for investigation and treatment of infertility. She was first seen at the age of 19 years with a complaint of primary amenorrhea, having presented without any secondary characteristics and anosmia. She had very low serum gonadotropin and estrogen levels, and inadequate responses to repeated gonadotropinreleasing hormone (GnRH) tests. In addition, olfactory magnetic resonance imaging (MRI) revealed olfactory tract agenesis. She was diagnosed with Kallmann’s syndrome and

Role of increased estradiol on altering the follicle diameters and gonadotropin concentrations that have been reported for double-ovulating heifers

During the preovulatory period in heifers that ovulate from two compared to one follicle, circulating concentrations of estradiol-17β (E2) are greater, diameter of follicles and concentration of FSH are reduced, and the LH surge occurs sooner. The effect of increased E2 on the reported characteristics of double ovulation was studied by treating heifers with 0.07 mg E2, 0.09 mg E2, or vehicle in four treatments at 6-h intervals ( n = 6 heifers/group), beginning at the time of expected follicle deviation (largest follicle, 8.5 mm). There were no significant differences on follicle diameters or hormone concentrations between the 0.07 and 0.09 mg E2 groups, and heifers were combined into one E2 group ( n = 12). The E2 treatments induced concomitant preovulatory surges in LH and FSH at 34.0 ± 2.6 h after first treatment, compared to 57.6 ± 4.5 h in the vehicle group ( P < 0.0002). The E2 treatments did not affect FSH concentrations during the preovulatory gonadotropin surge. The diameter of the preovulatory follicle at the LH peak was smaller ( P < 0.0001) in the E2-treated group (10.2 ± 0.2 mm) than in the vehicle group (13.1 ± 0.6 mm). The hypothesis was not supported that the previously reported increase in circulating E2 in heifers with double preovulatory follicles accounts for the reported lesser concentrations in the preovulatory FSH surge in heifers with double ovulations. Hypotheses were supported that the reported earlier occurrence of the preovulatory LH surge and smaller preovulatory follicles in heifers with double ovulations are attributable to the reported increase in E2 from the double preovulatory follicles.

Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation)

The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Thoroughbred mares, aged 2–26 years, were examined with ultrasound to ascertain ovulation date to within 24h, and pre-ovulatory follicle(s) (F1) diameter. Mares were divided into groups according to age (7 groups, 2–4 yr, 5–7 yr, 8–10 yr, 11–13 yr, 14–16 yr, 17–19 yr, >19 yr), time within the season (16 half-month groups, from Feb 1 st to Sept 30 th), and pre-ovulatory follicles (single, {SO} or multiple {MO}). Overall average F1 diameter was 39.95 ± 4.84 mm (range 22–50 mm). Mare age had a significant (P < 0.001) negative effect on F1 diameter (largest F1 38.95 ± 5.61 mm, mares 2–4 yrs; smallest F1 33.30 ± 4.66 mm, mares >19 yrs) as did season (largest F1 44.20 ± 3.95 mm, Feb 1 st–14 th; smallest F1 33.74 ± 4.87 mm, Aug 15 th–31 st) and the presence of more than one pre-ovulatory follicle (MO F1 35.45 ± 4.53 mm; SO F1 37.44 ± 4.84 mm). In conclusion older mares, bred towards the end of the breeding season, especially if MO were present, were more likely to ovulate from smaller follicles. If, as suggested, small pre-ovulatory follicle size is associated with low oocyte viability, then this may account, at least in part, for the poor fertility rates characteristic of older MO mares, bred later in the season and so justify increased monitoring and careful reproductive management of such mares.

Effect of Direct Ovarian Infusion of Bone Morphogenetic Protein 6 (BMP6) on Ovarian Function in Sheep1

Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 microg/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 +/- 2.8 h in treated vs. 53.3 +/- 2.7 h in controls; P < 0.05) and smaller preovulatory follicles (4.1 +/- 0.2 mm in treated vs. 5.3 +/- 0.1 mm in controls; P < 0.01), which gave rise to smaller corpora lutea (9.5 +/- 0.8 mm in treated vs. 11.7 +/- 0.6 mm in controls; P < 0.05). There was, however, no effect of infusion on ovulation rate. Despite the changes in the size of the ovulatory follicles, when the hormonal data were aligned to the time of the luteinizing hormone surge, there were no differences in preovulatory estradiol, androstenedione, or inhibin A between groups. This study therefore provides strong in vivo evidence to support the hypothesis that BMP6 is an important local regulator of ovarian function and that alterations in BMP6 cellular signaling may explain some of the effects of the FecB mutation in inducing precocious maturation of ovulatory follicles.