Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

Embryo production in superstimulated llamas pre-treated to inhibit follicular growth

Llamas are monotocous and the length of their gestation period varies between 342 and 350 days. Thus the average number of offspring any female can produce throughout her reproductive life is very limited to spread a desired genome. The multiple ovulation and embryo transfer (MOET) technique allows an alternative to this limitation and reduces the generation interval. The objective of this study was to evaluate embryo recovery in superstimulated llamas which had previously been hormone-treated to inhibit follicular growth. A total of 50 female llamas were monitored daily via rectal palpation and ultrasound and divided according to their ovarian follicular growth into four phases. The females in each phase were then randomly divided into two groups: A (n = 20) received a single dose of 1 mg of estradiol benzoate (EB) on the first day of the treatment + 100 mg of progesterone (P4) i.m. for 5 days with 5 animals per phase and B (n = 20) received 1 mg EB at onset + 150 mg P4 i.m. for a period of 5 days with 5 animals per phase. Group C (n = 10) or control did not receive any prior hormonal treatment and the females were in follicular phase I. All groups were monitored daily and, in the presence of ovarian follicles smaller than the dominant size at the end of treatment, all were superstimulated with 1000 IU eCG. For plasma progesterone concentration recording, daily blood samples were collected from days ?1 to 5 in the treated females in Group A and B. No significant differences were observed regarding the inhibition of follicle growth and in the plasma progesterone concentrations between Group A and B. The ovarian response to superstimulation was 56.2%, 71.4% and 90%, with the average number of dominant follicles produced per female being 4.4 ± 0.9; 4.8 ± 0.7 and 4.6 ± 0.6 in Groups A, B and C, respectively. The embryo recovery rate was 77.7%; 90% and 66.7% and the average number of embryos recovered per female was 2.9 ± 0.9; 2.6 ± 0.9 and 2.4 ± 0.8 for Groups A, B and C, respectively. In Groups A and B, the static follicular phase (III) seemed to be ideal for initiating the assisted reproductive technique of MOET. Although prior administration of P4 + EB seems to have no effect on the number of females that responded to the superstimulation treatments, the number of embryos recovered showed a tendency to be higher when ovarian follicle growth inhibition was performed beforehand.     

Effects of estradiol and progesterone on circulating LH and FSH secretion,and ovarian antral follicle growth in anestrous ewes

In the ewe, ovarian antral follicles emerge or grow in a wave-like pattern and each wave is preceded by a peak in the serum FSH level. The purpose of the current study was to investigate whether in anestrous Western White Face ewes, a combination of progesterone and estradiol affects the circulating FSH peak secretion and the number of small ovarian follicles. Five ewes were treated with subcutaneous silastic rubber implants (10 cm × 0.47 cm), containing 10% estradiol-17β w/w (controls) and 5 ewes were treated with the same estradiol implant, along with subcutaneous implants (11 cm × 0.48 cm) containing 10% progesterone w/w for 12 days. Daily transrectal ovarian ultrasonography and blood sampling was performed from 5 days before, to 9 days after the period of implantation. Blood samples were also taken every 12 min for a 6 h period on day −2, 6 and 13 prior to or after implant insertion (day 0, day of implant insertion). Pulsatility in the serum LH levels was eliminated by the implants (P < 0.05). During the implantation period, the serum FSH peak amplitude was lower in ewes treated with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol (P < 0.05). No follicular waves emerged during implant treatment in both groups (P < 0.05) and the number of serum FSH peaks did not differ during implantation, compared to before implantation. During the implantation period, the number of small follicles did not differ in ewes with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol. To conclude, supra-physiological concentrations of estradiol completely eliminated the serum LH pulsatality and suppressed the follicular wave emergence, while the FSH secretory peaks that preceded the follicular waves were not affected. Supra-physiological concentrations of estradiol-17β with physiological concentrations of progesterone decreased the serum FSH peak amplitude, eliminated the serum LH pulses, but did not decrease the size of the small follicle pool in anestrous ewes.     

The effect of the absence or presence of a corpus luteum on the ovarian follicular population and serum oestradiol concentrations during the estrous cycle in Sanjabi ewes

The aim of this study was to determine if the presence or absence of a corpus luteum (CL) during estrous synchronization in ewes can affect the ovarian follicular population and the serum oestradiol concentrations. The estrous cycles of 197 Sanjabi ewes were synchronized using a 12-day treatment with intravaginal progestagen sponges (Chronogest^®). Estrus was detected in 144 ewes, 27–39 h after sponge removal. Blood samples were taken daily from day 2 and continued for 19 days and analyzed for serum oestradiol concentration. Nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovaries per ewe were classified as those without, or with one or two CL's, for each slaughter day. Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) very small (<2 mm), (ii) small (2–3.4 mm), (iii) medium (3.5–5 mm) and (iv) large (>5 mm) categories, and the respective numbers recorded. Results indicated, the number of ovarian follicles to decrease (P < 0.01) from days 1 to 5 of the cycle and showed a significant increase on day 7. Numbers were high again on day 11 and decreased (P < 0.01) on day 16 of the estrous cycle. The serum oestradiol concentrations were significantly higher (P < 0.001) in the double than in the single ovulating animals (one or two CL's, respectively) on days 2–0. However serum levels were also significantly higher (P < 0.001) in single, than twin ovulating animals on days 4–5 and 12–16 of the estrous cycle. There were no significant differences in the total number of very small follicles between animals without and those with two CL's. The number of small, medium and large follicles in ewes, with or without a CL on the ovary was significantly higher (P < 0.01) than ewes with two ovulations at certain stages of the estrous cycle. The present study provides evidence of differences in the follicular ovarian population in ovaries without CL's and double ovulations. The existence of an intraovarian effect of the CL numbers on follicular population is demonstrated.     

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7 d, followed by the administration of 150 μg d-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment 1 (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.     

Sex differences in jaw muscle duty factors during exercise in two environments: A pilot study

It is unknown if females and males use jaw muscles similarly during exercise. This pilot study assessed jaw elevator muscle duty factors (DFs = time of muscle activity/total recording time) at repeated sessions to test if DFs are reliable and different between sexes during exercises in two environments. Ten female and seven male subjects recruited from university soccer teams provided informed consent. Surface electromyography was recorded from masseter and temporalis muscles during biting and leg-extension laboratory exercises. Average activities to produce 20 N bite-forces for each muscle and subject determined thresholds (5–80%·T20 N) for subject-specific DF calculations during exercises performed in laboratory and natural environments. Subjects self-recorded via portable electromyography equipment during in-field leg-extension and weight-lifting exercises. Effects of variables on DFs were assessed via ANOVA (α = 0.05) and simple effects testing (Bonferroni-adjusted p ⩽ 0.012). All subjects used jaw muscles during exercises in both environments. DFs between laboratory sessions were reliable (R = 0.84). During laboratory exercises, male temporalis DFs were significantly higher than female DFs from both muscles (p ⩽ 0.001). During in-field exercises females had higher DFs during weight-lifting while males had higher DFs during leg-extensions. In-field sex differences were significant at most thresholds and showed larger effect sizes for leg-extension compared to weight-lifting exercises.     

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O₂) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (≥150 μm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O₂ concentrations (5% and 20% O₂). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O₂ concentrations (P < 0.05). When the O₂ tensions were compared to each other in the different days of culture, 20% O₂ was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O₂ (P < 0.05). However, follicles cultured with 5% O₂ had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O₂ (P < 0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O₂ tensions. However, only oocytes (16.7%) from follicles cultured in 20% O₂ resumed meiosis. In conclusion, concentration of 20% O₂ was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.     

Glycomic analyses of ovarian follicles during development and atresia

To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n = 6) and atretic (n = 6) follicles in each of three size ranges: small (3–5 mm), medium (6-9 mm) and large (10 mm or more) (n = 29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n = 14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ?uronic acid with 4-sulfated N-acetylgalactosamine and ?uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan.     

Ultrasonographic imaging of the reproductive tract and surgical recovery of oocytes in Cebus apella (capuchin monkeys)

Two-dimensional real-time and Doppler ultrasonography are valuable non-invasive methods to assess reproductive anatomy and physiology. In adult, postpubertal female Cebus apella (capuchin monkeys), the objectives were to determine (1) uterine and ovarian dimensions, ovarian follicular dynamics, day of ovulation, and arterial blood flow of uterus and utero-ovarian ligament during the follicular phase of the menstrual cycle and (2) the number of oocytes aspirated from antral follicles at laparotomy. Based on two-dimensional, transabdominal B-mode ultrasonography, mean (± S.E.M.) length, height, width, and volume of the uterus were 17.9 ± 0.4, 12.4 ± 0.3, 13.6 ± 0.3 mm, and 1.55 ± 0.08 mL, respectively, and of the ovary were 13.4 ± 0.2, 8.2 ± 0.1, 7.7 ± 0.1 mm, and 4.5 ± 0.2 mL. Ovarian follicles were monitored for 6 days before ovulation, which occurred on day 9.3 ± 0.5 (range, days 7–11; day 1 = start of menses), with 10 of 12 ovulations in the right ovary. Diameter and volume of the preovulatory follicle were 10.1 ± 0.2 mm and 0.55 ± 0.03 mL (on the estimated day of ovulation) and of the CL were 8.1 ± 0.4 mm and 0.3 ± 0.05 mL. Resistivity and pulsatility indices were 0.86 ± 0.02 and 2.15 ± 0.11 for uterine arteries, and were 0.69 ± 0.04 and 1.63 ± 0.15 for the utero-ovarian ligament (UOL) artery; just prior to ovulation, both indices peaked (P < 0.05) in the uterine artery ipsilateral to the side of ovulation, but both reached a nadir (P < 0.05) in the UOL artery. In the absence of ovarian stimulation, 31 oocytes (diameter, 137 ± 10 μm) were aspirated (average of 2 oocytes/(female attempt)) on days 5, 7, and 9. In conclusion, transabdominal ultrasonography facilitated assessment of reproductive anatomy and physiology in C. apella adult females. Resistance and pulsatility indices of uterine and UOL arteries changed near the time of ovulation. Dominant follicles were easiest to aspirate at 8–9 mm in diameter (∼day 9), with intact cumulus-oocyte complexes recovered from ovarian follicles 2–9 mm in diameter.     

Oestrus cycle characteristics and prediction of ovulation in Catalonian jennies

The Catalonian donkey breed is in danger of extinction, and much needs to be learned about the reproductive features of its females if breeding and conservation programmes are to be successful. This study reports the oestrous behaviour, oestrus cycle characteristics and dynamic ovarian events witnessed during 50 oestrous cycles (involving 106 ovulations) in 10 Catalonian jennies between March 2002 and January 2005. These jennies were teased, palpated transrectally and examined by ultrasound using a 5 MHz linear transducer—daily during oestrus and every other day during dioestrus. Predictors of ovulation were sought among the variables recorded.The most evident signs of oestrus were mouth clapping (the frequent vertical opening and closing of the mouth with ears depressed against the extended neck) and occasional urinating and winking of the vulval lips (homotypical behaviour). Interactions between jennies in oestrus were also recorded, including mounting, herding/chasing, the Flehmen response, and vocalization (heterotypical behaviour).Nine jennies ovulated regularly throughout the year; one had two anovulatory periods (54 and 35 days). The length of the oestrus cycle was 24.90 ± 0.26 days, with oestrus itself lasting 5.64 ± 0.20 days (mean ± S.E.M.) and dioestrus 19.83 ± 0.36 days. The incidence of single, double and triple ovulations was 55.66% (n = 59), 42.45% (n = 45) and 1.89% (n = 2), respectively. No significant difference was seen in the number of ovulations involving the left and right ovaries (52.63% [n = 70] compared to 47.37% [n = 63] respectively; P > 0.05). The mean interval between double ovulation was 1.44 ± 3.98 days. The mean diameter of the preovulatory follicle at day −1 was 44.9 ± 0.5 mm; the mean growth rate over the 5 days before ovulation was 3.7 mm/day.Data on preovulatory changes in oestrous behaviour, follicle size, follicle texture, the echographic appearance of the follicle and uterus, and uterine tone were subjected to stepwise logistic regression analysis to detect predictors of ovulation. The logit function showed the best predictors to be follicle size, follicular texture and oestrous behaviour. Certain combinations of these three variables allow the prediction of ovulation within 24 h with a probability of >75%.     

Study of folliculogenesis in vivo in guinea pig

Understanding normal folliculogenesis in guinea pigs is fundamental as a first step towards the development of a guinea pig follicle culture system. The aims of this study were (1) to characterise morphological changes during follicular development in vivo and (2) to describe the growth pattern of follicles. Cycling guinea pigs were infused with 5-bromo-2′-deoxyuridine for 1 or 2 weeks and sacrificed at time points ranging from 0 to 37 days after the infusion. The granulosa cell number in the largest cross-sections increased from 25.0 ± 6.1 (mean ± S.D.) in primary (type 2) to 192.0 ± 65.9 in preantral (type 5) and 256.3 ± 96.9 in antral (type 6) follicles. The oocyte diameter increased from 44.8 ± 6.2 μm (type 2) to 72.8 ± 9.1 μm (type 5) and 78.9 ± 9.3 μm (type 6) and the follicle diameter from 67.9 ± 10.1 μm (type 2) to 188.9 ± 29.7 μm (type 5) and 231.0 ± 56.1 μm (type 6). After a 1-week labelling period, about 71% of type 2 follicles had at least one labelled granulosa cell, as did 95% of type 3–4, and 100% of type 5 and 6. About 1 week was needed to achieve 95% mitotic activity in granulosa cells (GC) of type 5 and 6 follicles, while about 2 weeks was required to achieve 100% mitotic activity in GC of type 3–4 and more than 2 weeks for GC of type 2 follicles. These data provide some baselines for the examination of a guinea pig follicle culture system.     

Effects of testosterone and 2-hydroxyflutamide on progesterone receptor expression in porcine ovarian follicles in vitro

The purpose of the study was to test the possible role of the androgen receptor (AR) agonist (testosterone; T), an AR antagonist (2-hydroxyflutamide; 2-Hf) or combination of both (T + 2-Hf) on progesterone receptor (PGR) expression in cultured porcine granulosa cells (GCs) or whole follicles. GCs isolated from mature pig follicles (6–8 mm in diameter) were cultured for 48 h. Experimental cultures were carried out with the addition of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-Hf for the last 24 h of culture. To better imitate in vivo conditions, isolated whole porcine follicles (6–8 mm in diameter) were cultured for 24 h in an organ culture system, with the addition of the same factors. The cells or sections obtained from cultured follicles were processed for PGR immunocytochemical or immuno-histochemical staining. In addition, expression of PGR protein was determined by Western blot and progesterone (P₄) concentrations in the culture media were measured by a radioimmunoassay. We found that isoform A of PGR is expressed in both granulosal and follicular cultures. The 2-Hf in the presence of T increased PGR protein expression in porcine GCs and whole follicles. In both granulosal and follicular cultures, 2-Hf or T alone inhibited P₄ secretion, but simultaneous addition of 2-Hf and T increased P₄ secretion. Our results indicate that androgens may be involved in the control of PGR expression in porcine GCs in vitro. Moreover, we suggest a potential auto/paracrine regulation of the follicular function by androgen-dependent signaling pathway.     

Differences in preovulatory follicle dynamics and timing of preovulatory LH surge affect fertility of maiden sheep reared in semi-arid extensive conditions

In the current study follicular dynamics, pituitary function, ovulatory response and luteal activity of 30 maiden Barbarine sheep were analyzed according to oestrus occurrence and lambing outcome after oestrus synchronisation with cloprostenol. Animals were retrospectively classified in three groups named as O? (n = 7, ewes not displaying oestrus), O+L? (n = 7, ewes showing oestrus but failing to lamb) and O+L+ (n = 16; ewes showing oestrus and lambing thereafter). All the sheep ovulated and daily transrectal ultrasonographies revealed that preovulatory follicles were present at cloprostenol injection in all the animals. In sheep O+L+ and O+L?, 50% and 57% of the ovulatory follicles were the largest follicles at cloprostenol treatment (mean size of 4.1 ± 0.26 mm and 4.3 ± 0.74 mm, respectively). In O? ewes, the same percentage was higher (86%, P < 0.05 when compared to group O+L+; mean size of 4.0 ± 0.46 mm). The number of large follicles and the final diameter of the ovulatory follicles at oestrous tended thereafter to be higher in group O+L+ (1.4 ± 0.1 and 6.4 ± 0.2) than in groups O+L? (1 ± 0.2 and 5.7 ± 0.36) and O? (0.9 ± 0.2 and 5.9 ± 0.5, respectively). Conversely, the number of medium follicles at oestrus detection was higher in the group O+L? (2.1 ± 0.3, P < 0.05) than in the other two groups (1 ± 0.2 and 1 ± 0.3 for O+L+ and O? respectively). Timing of preovulatory LH surge was earlier for ewes O? (24.0 ± 4.75, P < 0.05) than for sheep O+L+ and O+L? (37.9 ± 2.45 h and 38.0 ± 4.75 h, respectively) and 94% of O+L+ ewes had a LH surge between 16 h and 64 h after cloprostenol injection compared to 57% in O+L? and O? groups (P < 0.05). Thus, maiden Barbarine sheep failing to display oestrus or conceive showed alterations in their follicular dynamics and, thereafter, pituitary function and ovulatory response.     

Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus)

The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat's estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period (day 0=day of sponges withdrawal), 2 of the goats were bilaterally ovariectomized. Follicles with a diameter >1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5±9.8 h. LH peak concentration averaged 44±5.3 ng/ml and the mean length of the preovulatory surge (amounts over 10 ng/ml) was 8.9±0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles (58 vs. 30, P<0.05). The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles (23 vs. 6, P<0.05). On day 2, the number of advanced atretic follicles was greater than early atretic follicles (10 vs. 4, P<0.05). There was an increase in the number of early atretic follicles from day 2 to day 4 (4 vs. 9, P<0.05), which was consistent with the effects of the preovulatory LH surge.     

Correlations between ovarian follicular blood flow and superovulatory responses in ewes

The primary goal of this study was to employ ultrasonography to examine the ovaries of ewes undergoing superovulatory treatment for correlations between antral follicular blood flow and ovarian responses/embryo yields. Five Santa Inês ewes were subjected to a short- (Days 0–6, Group 1) and five to a long-term progesterone-based protocol (Days 0–12, Group 2) to synchronize estrus and ovulations after the superovulatory treatment. Porcine FSH (pFSH, 200 mg) was administered in 8 decreasing doses over 4 days, starting on Days 4 and 10 in Groups 1 and 2, respectively. After CIDR removal, all ewes were bred by a ram and embryos were recovered surgically 7 days later. Transrectal ovarian ultrasonography was performed the day before and on all 4 days of the superovulatory treatment. Both an arbitrary-scale [(0) non-detectable; (1) small; (2) moderate; (3) intense blood flow] and quantitative analysis of the blood flow area were used to assess the follicular blood flow in color Doppler images. There were no significant correlations between the arbitrary blood flow scores and superovulatory responses in the ewes of the present study. However, there was a positive correlation between the quantitative estimates of follicular blood flow on the final day of the superovulatory treatment, and the number (DA: r = 0.68, P < 0.05; DA/TA × 100%: r = 0.85, P < 0.05) and percentage (DA: r = 0.65, P < 0.05; DA/TA × 100%: r = 0.91, P < 0.001) of unfertilized eggs (DA: Doppler area, TA: total area of the largest ovarian cross section). This experiment presents a commercially practical tool for predicting superovulatory outcomes in ewes and evidence for the existence of follicular blood flow threshold that may impinge negatively on oocyte quality when surpassed during hormonal ovarian superstimulation.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Biochemical changes in the follicular fluid of the dominant follicle of high producing dairy cows exposed to heat stress early post-partum

High yielding dairy cows experience a negative energy balance (NEB) early post-partum and it was hypothesized that this may be aggravated under summer heat stress (HS) conditions. In this study, which was performed in Egypt, 20 Holstein cows were followed during summer (n = 10) and winter (n = 10) seasons. All cows were multiparous and kept at the same herd. Blood was sampled from each cow starting 1 week before the expected calving date and then at 1-week intervals until week 6 post-partum. From week 2 to 6 post-partum follicular fluid was collected through transvaginal follicular fluid aspiration at 6 days intervals. Ambient air temperature (AT) and relative humidity (RH) were recorded and temperature–humidity index (THI) was calculated as well. Respiration rate (RR), rectal temperature (RT), and body condition score (BCS) were recorded for each cow at the time of blood sampling. Concentrations of glucose, insulin like growth factor-1 (IGF-1), non-esterified fatty acids (NEFA), urea and total cholesterol (TC) were measured in each blood and follicular fluid sample. All the cows showed a significantly higher RR and RT in summer (95.5 ± 1.1 and 39.88 ± 0.06, respectively) than in winter (43.89 ± 0.61 and 38.94 ± 0.07, respectively) (P < 0.001). Body condition score loss during the early post-partum period was higher in summer than in winter (1.1 ± 0.07 vs. 0.85 ± 0.06 point, respectively) (P < 0.001). The average dominant follicle diameter was significantly lower in summer than in winter during the period of negative energy balance (11.6 ± 0.7 mm vs. 15.3 ± 1.2 mm, respectively) (P < 0.01). Under summer heat stress, the concentrations of glucose (2.98 ± 0.07 and 2.19 ± 0.04 mmol/L), IGF-1 (106.7 ± 2.9 and 99.0 ± 3.4 ng/ml) and TC (137.3 ± 5.3 and 62.2 ± 5.1 mg/dl) in blood and FF, respectively, were significantly lower than winter concentrations by (0.17 ± 0.03 mmol/L, P < 0.001 and 0.26 ± 0.06 mmol/L, P < 0.001), (12.3 ± 3.6 ng/ml, P < 0.001 and 9.0 ± 2.7 ng/ml, P < 0.001) and (20.7 ± 1.8 mg/dl, P < 0.001 and 7.3 ± 1.1 mg/dl, P < 0.01), respectively. However, the concentrations of NEFA (0.68 ± 0.14 and 0.22 ± 0.02 mmol/L) and urea (9.27 ± 0.34 and 9.96 ± 0.25 mmol/L) in blood and FF, respectively, were significantly higher in summer compared to winter (0.50 ± 0.08 mmol/L, P < 0.001 and 0.20 ± 0.02 mmol/L, P < 0.001) and (8.77 ± 0.23 mmol/L, P < 0.05 and 8.96 ± 0.29 mmol/L, P < 0.001), respectively, throughout the experimental period. The results of the present study indicate that heat stress early post-partum aggravates NEB in high yielding dairy cows, reduces BCS, dominant follicle diameter and alters the biochemical concentrations in the follicular fluid of the dominant follicle which may result in inferior oocyte and granulosa cell quality and hence poorer fertility.     

The effects of EGF and IGF-1 on FSH-mediated in vitro maturation of domestic cat oocytes derived from follicular and luteal stages

The objective of this study was to evaluate the influence of epidermal growth factor (EGF) and insulin like growth factor-I (IGF-1) on the in vitro maturation of cat oocytes recovered from follicular and luteal stage ovaries. Oocytes from follicular (n = 580) and luteal (n = 209) stages were harvested and divided into four groups, which were cultured in FSH-mediated maturation medium supplemented with: (1) EGF alone (25 ng/mL); (2) IGF-1 alone (100 ng/mL); (3) EGF + IGF-1 (25 ng/mL EGF + 100 ng/mL IGF-I); or (4) no growth factor (control). The proportion of follicular stage oocytes reaching the metaphase II stage was significantly higher than that of oocytes obtained at the luteal stage in both control and study groups (p < 0.001). The percentages of oocytes reaching the metaphase II stage during the follicular period were 62.6% in control; 70.9% in EGF; 72.8% in IGF-1, and 78.1% in EGF + IGF-1 groups, whereas the respective values for gametes collected from luteal stage ovaries were 12.5%, 17.5%, 12.5%, and 16.9%. Additionally, the differences between the study and control groups were significant in the case of follicular stage oocytes. Finally, supplementing the maturation medium with EGF and/or IGF-1 significantly enhanced the meiotic maturation of oocytes recovered from follicular stage ovaries. The present study also demonstrated that the combination of EGF and IGF-I provides an additional or synergic effect on meiotic maturation of oocytes recovered from the follicular stage.     

Ovarian response and pregnancy rate following different doses of eCG treatment in Chall ewes

The purpose of the study was to investigate the effects of different doses of equine chorionic gonadotropin (eCG) treatment on follicular development, ovulation and pregnancy rate during the breeding season in fat-tailed Chall ewes. Seventy-two cycling (62.5 ± 2.5 kg), multiparous Iranian Chall ewes were used in the trial. The ewes were randomly allocated to 6 groups (n = 12/group). Estrus was synchronized with the aid of controlled intravaginal drug release (CIDR) devices, inserted for 14 days. At the time of CIDR removal (day 14), the ewes received i.m. either 0 (control group, G0), 450 (G450), 550 (G550), 650 (G650), 750 (G750) or 850 (G850) IU eCG. Vasectomized rams were used to detect estrus in the ewes from 24 h after CIDR removal. Ovarian follicular activity was monitored with the aid of transrectal ultrasonography on the day of CIDR insertion (day 0) and daily from the day of eCG treatment (day 14), until estrus (day 16). During these days, blood samples were collected for the determination of plasma progesterone and estradiol concentrations. Laparoscopic intrauterine inseminations were conducted 54–60 h after CIDR removal. The number of CL's and pregnancy diagnosis was recorded using ultrasonography 7 and 54 days following AI, respectively. Half of ewes in control group and most of the ewes treated with eCG showed signs of estrus within 36 h of CIDR removal. The ewes in groups G750 and G850 recorded the highest number of large follicles at estrus and CL's 7 days later. The pregnancy rate in groups G550 (75.0%) and G650 (75.0%) was higher (P < 0.05) than that in the other groups. The ovarian response and estradiol concentration, as well as pregnancy rate showed that 550 or 650 IU eCG treatment is the most effective doses in improving the pregnancy rate in Iranian Chall ewes.