In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

Folliculogenesis-related genes are differently expressed in secondary and tertiary ovarian follicles.

The relative mRNA abundance of 10 genes associated with folliculogenesis was compared between late preantral (secondary) and early antral (tertiary) ovarian follicles in goats. In total, 100 follicles in each category were mechanically isolated. The relative transcript abundance of the mRNAs were determined by qPCR. Data were analyzed using unpaired Student's t-test. Of the 10 tested genes, ABLIM mRNA was not detected in either follicle category, six genes (SLIT3, TYMS, GTPBP1, AKR1C4, PIK3R6, and MAOB) were upregulated in secondary follicles compared with tertiary follicles, and three genes (ARHGEF12, CLEC6A, and CYTL1) showed similar mRNA abundances in both secondary and tertiary follicles. In conclusion, SLIT3, GTPBP1, AKR1C4, and PIK3R6 mRNA abundance was upregulated in secondary follicles (preantral phase) compared with in tertiary follicles (antral phase) in goats.

The mule (Equus mulus) as a recipient of horse (Equus caballus) embryos: Comparative aspects of early pregnancy with mares.

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos.

Novel prospects for evaluation of follicle wall blood flow using color-Dopplerultrasonography

The goal of this study was to develop anobjective method for evaluation of ovarian follicle wallblood flow in cattle. Two subjective methods were used:(I) real-time ultrasound evaluations performed by oneoperator in the barn and (II) video clip evaluationsperformed by four operators in the laboratory. Thefollowing objective methods evaluated in the laboratorywere used for comparison: (I) percentage of follicle wallcircumference under blood flow (WUF) and (II) pixelarea of color-Doppler signals. Cows (n = 21) weresubmitted to a synchronization protocol, follicles ≥7 mmwere measured, and blood flow was evaluated every 12 huntil ovulation using color-Doppler ultrasonography. Nodifference (P > 0.05) was observed among laboratoryoperators from day 2 of training onwards. Therefore,an average score of all operators was used forcomparisons among different methods. Both subjectiveand objective methods of evaluation showed anincrease (P < 0.0001) in follicle blood flow over time.Higher (P < 0.001) correlations were obtained betweenWUF and subjective laboratory evaluation thanbetween WUF and pixel area or WUF and subjectivebarn data. Higher (P < 0.0003) correlation coefficientswere observed for WUF than for the pixel area whencompared with the barn (r = 0.70 vs. r = 0.42) orlaboratory (r = 0.84 vs. r = 0.62) data. Subjectiveevaluations at the laboratory and barn producedstronger correlations with WUF (P < 0.0008) than withpixel area (P < 0.01). In conclusion, WUF is aneffective and reliable method for objective evaluationof follicle wall blood flow in cows.(AU)

Novel prospects for evaluation of follicle wall blood flow using color-Dopplerultrasonography

The goal of this study was to develop anobjective method for evaluation of ovarian follicle wallblood flow in cattle. Two subjective methods were used:(I) real-time ultrasound evaluations performed by oneoperator in the barn and (II) video clip evaluationsperformed by four operators in the laboratory. Thefollowing objective methods evaluated in the laboratorywere used for comparison: (I) percentage of follicle wallcircumference under blood flow (WUF) and (II) pixelarea of color-Doppler signals. Cows (n = 21) weresubmitted to a synchronization protocol, follicles ≥7 mmwere measured, and blood flow was evaluated every 12 huntil ovulation using color-Doppler ultrasonography. Nodifference (P > 0.05) was observed among laboratoryoperators from day 2 of training onwards. Therefore,an average score of all operators was used forcomparisons among different methods. Both subjectiveand objective methods of evaluation showed anincrease (P < 0.0001) in follicle blood flow over time.Higher (P < 0.001) correlations were obtained betweenWUF and subjective laboratory evaluation thanbetween WUF and pixel area or WUF and subjectivebarn data. Higher (P < 0.0003) correlation coefficientswere observed for WUF than for the pixel area whencompared with the barn (r = 0.70 vs. r = 0.42) orlaboratory (r = 0.84 vs. r = 0.62) data. Subjectiveevaluations at the laboratory and barn producedstronger correlations with WUF (P < 0.0008) than withpixel area (P < 0.01). In conclusion, WUF is aneffective and reliable method for objective evaluationof follicle wall blood flow in cows.

Relationships between follicle and corpus luteum diameter, blood flow, and progesterone production in beef cows and heifers: preliminary results

The aim of the present study in beef cattle was to investigate potential differences in follicle size and follicle wall-blood flow between cows and heifers and to compare follicle wall-blood flow between smaller and larger follicles. Cows and heifers were treated with a synchronization protocol and follicles and CLs were measured and evaluated for blood flow. Pregnancy diagnosis was performed on day 50 of the protocol. Cows had larger (P < 0.008) follicles than heifers. Cows, heifers, and pregnant and non-pregnant cows did not differ (P > 0.05) in CL diameter, CL blood flow, and plasma progesterone concentrations. Moderate correlations between follicle diameter and follicle blood flow were observed for cows (r = 0.51; P < 0.002) and heifers (r = 0.61; P < 0.0001). Pregnant cows tended (P < 0.1) to have larger follicles between 12 to 60 h before ovulation, and had larger (P < 0.05) follicles than non-pregnant cows at hour 24 before ovulation and at hour 12 before maximum values. Pregnant cows had greater (P < 0.05) follicle blood flow than non-pregnant cows at hours −36 and −24 before maximum values. Follicle blood flow was greater (P < 0.002) in the large follicles compared with the small follicles, and tended (P < 0.06) to be greater than in medium follicles. Moderate to strong correlations were found between follicle blood flow and diameter of small (r = 0.59; P < 0.002), medium (r = 0.50; P < 0.02), and large (r = 0.71; P < 0.0001) follicles. Pregnancy rates were similar (P > 0.05) among all follicle diameter categories. In conclusion, synchronized beef cows and pregnant cows had larger follicles and greater blood flow than heifers and non-pregnant cows, and follicle wall blood flow was closely associated with increasing follicle diameter.(AU)

Relationships between follicle and corpus luteum diameter, blood flow, and progesterone production in beef cows and heifers: preliminary results

The aim of the present study in beef cattle was to investigate potential differences in follicle size and follicle wall-blood flow between cows and heifers and to compare follicle wall-blood flow between smaller and larger follicles. Cows and heifers were treated with a synchronization protocol and follicles and CLs were measured and evaluated for blood flow. Pregnancy diagnosis was performed on day 50 of the protocol. Cows had larger (P 0.05) in CL diameter, CL blood flow, and plasma progesterone concentrations. Moderate correlations between follicle diameter and follicle blood flow were observed for cows (r = 0.51; P 0.05) among all follicle diameter categories. In conclusion, synchronized beef cows and pregnant cows had larger follicles and greater blood flow than heifers and non-pregnant cows, and follicle wall blood flow was closely associated with increasing follicle diameter.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 µg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 µg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth.

Number and density of equine preantral follicles in different ovarian histological section thicknesses.

Regardless of species, advances in preantral follicle culture and cryopreservation and transplant of ovarian tissue techniques are dependent on the number and density of preantral follicles in the ovary. This study tested the effect of different histological section thicknesses on number, classification, and density of equine preantral follicles. An ovarian fragment was obtained from 5- to 10-year-old mares (n = 14) after slaughter, and each fragment was submitted to three histological section thickness treatments: 3, 5, and 7 µm. The area (cm(2)) of each ovarian fragment was measured, and the sections were evaluated by light microscopy. The percentage of morphologically normal follicles (89%) was similar (P > 0.05) among primordial, transitional, and primary follicles and also among histological section thicknesses. A greater (P < 0.05) number of preantral follicles per histological section were seen in the 7-µm (8.0 ± 2.2) than that in the 3-µm (3.4 ± 0.7) treatment. Furthermore, a linear regression analysis reported that the number of preantral follicles increased (P < 0.05) when a thicker section treatment was used. However, no association (P > 0.05) between follicular density and treatment was observed. The mean number of preantral follicles per fragment (45.3 ± 18.8) and the follicular density (3.0 ± 0.5 follicles per cm(2)) were different (P < 0.05) among mares. In conclusion, this study on equine preantral follicles reported that (1) a 7-µm histological section thickness might be recommended because it allowed identification of a greater number of preantral follicles per sample, (2) a large individual variation in follicle population and density was detected regardless of histological section thickness, and (3) mares have a low number and density of preantral follicles when compared with those reported for other species.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

Equine preantral follicle harvesting, processing, and in vitro culture: the journey has already started

Preantral follicles are of great abundance in mammalian ovaries and the vast majority (>99.9%) never become ovulatory; therefore, the ability to rescue these otherwise wasted follicles seems very aooealing. Considering there are striking similarities in antral follicle dynamics between mares and women, the mare might become a good model to study early (preantral and antral) folliculogenesis in women, with several advantages related to using an animal model. Studies in our laboratory recently validated the use of a transvaginal ultrasound-guided ovarian. Biopsy Pick-Up (BPU) method to harvest preantral follicles using the mare as a model to study early folliculogenesis (Haag et al., 2013a, b, c). This article will review some of the important findings of our recent studies related to the harvesting, processing, and culture of equine preantral follicles and discuss those with the limited information availeble in the literature.

Equine preantral follicle harvesting, processing, and in vitro culture: the journey has already started

Preantral follicles are of great abundance in mammalian ovaries and the vast majority (>99.9%) never become ovulatory; therefore, the ability to rescue these otherwise wasted follicles seems very aooealing. Considering there are striking similarities in antral follicle dynamics between mares and women, the mare might become a good model to study early (preantral and antral) folliculogenesis in women, with several advantages related to using an animal model. Studies in our laboratory recently validated the use of a transvaginal ultrasound-guided ovarian. Biopsy Pick-Up (BPU) method to harvest preantral follicles using the mare as a model to study early folliculogenesis (Haag et al., 2013a, b, c). This article will review some of the important findings of our recent studies related to the harvesting, processing, and culture of equine preantral follicles and discuss those with the limited information availeble in the literature.(AU)

Effect of sequential medium on in vitro culture of goat ovarian cortical tissue.

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen.

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program.

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to -6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), -1 (68%), -2 (63%), -3 (66%), -4 (76%), -5 (61%), and -6 (27%). The -6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to -5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles.

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P<0.06) and cultured without FSH (P<0.1). There was a higher (P<0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P<0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P<0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P<0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.

Equine preovulatory follicle: blood flow changes, prediction of ovulation and fertility / Folículo pré-ovulatório equino: mudanças circulatórias, predição de ovulação e fertilidade

This review will focus on the main findings of our experiments that used B-mode and color-Doppler ultrasonography during the preovulatory period to study the morphological and blood flow/perfusion changes of the preovulatory follicle in mares. The topics to be addressed herein will be: ultrasonographic characteristics of the preovulatory follicle; B-mode echotextural changes of the follicle wall; blood flow and perfusion changes of the follicle wall; signs of impending ovulation; prediction of impending ovulation; types of preovulatory follicle outcomes such as ovulation, septated evacuation, hemorrhagic anovulatory follicle, and atresia; follicle blood flow during evacuation; early corpus luteum blood flow; and vascularity of the preovulatory follicle versus fertility.(AU)

This review will focus on the main findings of our experiments that used B-mode and color-Doppler ultrasonography during the preovulatory period to study the morphological and blood flow/perfusion changes of the preovulatory follicle in mares. The topics to be addressed herein will be: ultrasonographic characteristics of the preovulatory follicle; B-mode echotextural changes of the follicle wall; blood flow and perfusion changes of the follicle wall; signs of impending ovulation; prediction of impending ovulation; types of preovulatory follicle outcomes such as ovulation, septated evacuation, hemorrhagic anovulatory follicle, and atresia; follicle blood flow during evacuation; early corpus luteum blood flow; and vascularity of the preovulatory follicle versus fertility.(AU)

Equine preovulatory follicle: blood flow changes, prediction of ovulation and fertility / Folículo pré-ovulatório equino: mudanças circulatórias, predição de ovulação e fertilidade

This review will focus on the main findings of our experiments that used B-mode and color-Doppler ultrasonography during the preovulatory period to study the morphological and blood flow/perfusion changes of the preovulatory follicle in mares. The topics to be addressed herein will be: ultrasonographic characteristics of the preovulatory follicle; B-mode echotextural changes of the follicle wall; blood flow and perfusion changes of the follicle wall; signs of impending ovulation; prediction of impending ovulation; types of preovulatory follicle outcomes such as ovulation, septated evacuation, hemorrhagic anovulatory follicle, and atresia; follicle blood flow during evacuation; early corpus luteum blood flow; and vascularity of the preovulatory follicle versus fertility.

This review will focus on the main findings of our experiments that used B-mode and color-Doppler ultrasonography during the preovulatory period to study the morphological and blood flow/perfusion changes of the preovulatory follicle in mares. The topics to be addressed herein will be: ultrasonographic characteristics of the preovulatory follicle; B-mode echotextural changes of the follicle wall; blood flow and perfusion changes of the follicle wall; signs of impending ovulation; prediction of impending ovulation; types of preovulatory follicle outcomes such as ovulation, septated evacuation, hemorrhagic anovulatory follicle, and atresia; follicle blood flow during evacuation; early corpus luteum blood flow; and vascularity of the preovulatory follicle versus fertility.

Effect of ejaculation frequency and season on donkey jack semen.

Semen characteristics of first and second successive ejaculates from 6 jacks were evaluated weekly for 12 mo. The semen was collected at 4-h intervals, using an artificial vagina with a female in either natural or induced estrus. The statistical analysis was done by factorial delineation 2 x 2 in randomized blocks. Due to some ejaculation failures, the data had to be divided into 2 Groups (A and B) for statistical analysis: Group A - ejaculates preceded by 2 ejaculates in the previous week and Group B - ejaculates preceded by only 1 ejaculate in the previous week. If no statistical difference was observed between the groups in a given parameter, the data was grouped together. Semen characteristics for the first and second ejaculates, respectively, showed the following mean +/- SEM: gel-free semen volume 29.2 +/- 2.2 and 31.7 +/- 2.2 ml; progressive motility 71.0 +/- 1.6 and 72.9 +/- 1.6%; sperm vigor 3.8 +/- 0.1 and 4.1 +/- 0.1; live spermatozoa for Group A 82.6 +/- 2.1 and 82.3 +/- 2.1%, and for Group B 84.6 +/- 1.4 and 86.6 +/- 1.4%; total number of spermatozoa for Group A 10.6 +/- 0.8 x 10(9) and 5.8 +/- 0.8 x 10(9), and for Group B 13.3 +/- 1.2 x 10(9) and 9.6 +/- 1.2 x 10(9); head abnormalities for Group A 1.2 +/- 0.3 and 1.4 +/- 0.3%, and for Group B 1.6 +/- 0.3 and 1.9 +/- 0.3%; mid piece abnormalities 7.7 +/- 0.7 and 6.1 +/- 0.7%; tail abnormalities 7.3 +/- 0.7 and 6.8 +/- 0.7%; pH 7.6 +/- 0.0 and 7.6 +/- 0.0. Significant differences (P < 0.05) were observed between the animals for all sperm characteristics except for sperm vigor. The means for the first and second ejaculates were significantly different (P < 0.05) for the total number of spermatozoa in all the animals, while the percentage of mid piece abnormalities was significantly different in only 1 jack. Seasonal effects on sperm parameters were observed only for semen pH.