Antral follicle population in prepubertal and pubertal heifers.

The antral follicle count (AFC) is an important tool in the selection of bovine females destined for biotechnology. However, little is known about AFC in prepubertal and pubertal heifers. Some challenges inherent to the physiology of young females must be considered to achieve efficient rates with different procedures, such as ovum pick-up and IVF. This paper covers some important topics about ovarian physiology related to the population of antral follicles and reproductive efficiency in young female cattle.

Influence of follicle-stimulating hormone concentrations on the integrity and development of bovine follicles cultured in vitro.

SummaryThis study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin's solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

Molecular characteristics of granulosa and cumulus cells and oocyte competence in Nelore cows with low and high numbers of antral follicles.

The aims of this study were to investigate whether the number of antral follicles (AF) in the ovaries of Nelore cows is influenced with the developmental competence of oocytes to reach the blastocyst stage and to quantify the mRNA abundance of genes associated with folliculogenesis and oogenesis in granulosa and cumulus cells. A total of 168 cows were distributed into two experimental groups according to the number of AF, low (≤31) and high AF (≥92), which were determined based on the mean number of AF (61.14) ± SD (30.43). Granulosa and cumulus cells were used to assess the mRNA expression of 16 genes. Cumulus cells from cows with low AF had higher mRNA expression of genes involved in meiosis resumption (NPR-2, NPR-3) and cumulus cell expansion (FGF10), as well as a transcription factor involved in the regulation of oocyte maturation and cell proliferation (STAT3). Conversely, granulosa cells from females with high AF had higher expression of PGR and AMHR2a, which are involved in meiosis resumption and cumulus cell expansion. Cumulus-oocyte complexes (COCs) were collected from 356 cows with low and high AF populations to evaluate embryo development. Cleavage and blastocyst rates did not differ between the groups. In conclusion, our findings revealed that genes involved in folliculogenesis and oogenesis are differently expressed in cumulus and granulosa cells of cows having low and high numbers of AF. These molecular differences suggest that the regulation of oocyte maturation, meiotic resumption and cumulus expansion may be influenced by the number of AFs. However, the variations in gene expression were not associated with in vitro oocyte developmental competence to reach the blastocyst stage, which confirms that oocytes from Nelore cows with low and high numbers of AF are similarly able to mature, regulate the fertilization process and support pre-implantation embryo development.

The development and integrity of equine pre-antral follicles cultured in vitro with follicle-stimulating hormone (FSH) supplementation.

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre-antral follicles, the groups with 100 ng/ml FSH of 2-days culture as well as 50, 100 and 200 ng/ml FSH of 6-days culture provided the best results. In conclusion, the in vitro culture of abattoir-derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.

Pregnancy Rates to Fixed Embryo Transfer of Vitrified IVP Bos indicus, Bos taurus or Bos indicus × Bos taurus Embryos.

The pregnancy rates obtained after the transfer of cryopreserved in vitro-produced (IVP) embryos are usually low and/or inconsistent. The objective of this study was to evaluate the pregnancy rates of Holstein, Gyr and Holstein × Gyr cattle after the transfer of vitrified IVP embryos produced with X-sorted sperm. Seventy-two Gyr and 703 Holstein females were subjected to ovum pickup (OPU) sessions, followed by in vitro embryo production using semen from sires of the same breeds. Embryos (1636 Holstein, 241 Gyr and 1515 Holstein × Gyr) were exposed to forskolin for 48 h prior to vitrification. The pregnancy rate achieved with Gyr dam and sire was 46.1%, which was similar (p = 0.11) to that of Holstein dam and Gyr sire (40.3%). Crossing Gyr dams with Holstein sires resulted in a pregnancy rate of 38.9% and did not differ (p = 0.58) from the pregnancy rate obtained with the cross between Holstein dams and Gyr sires. The rate obtained with Holstein dam and sire was 32.5%. The average pregnancy rate was 36.6%, and no difference was found in the proportion of female foetuses (88.8%, in average) among breeds (p > 0.05). In conclusion, transfer of cryopreserved X-sorted embryos represents an interesting choice for dairy cattle. Despite the small differences between pregnancy rates, we highlight the efficiency of this strategy for all of the racial groups studied.

Comparison of antral and preantral ovarian follicle populations between Bos indicus and Bos indicus-taurus cows with high or low antral follicles counts.

The objective was to compare populations of antral and pre-antral ovarian follicles in Bos indicus and Bos indicus-taurus cows with high and low antral follicle counts. Nelore (Bos indicus, n = 20) and Nelore X Angus (1/2 Bos indicus-taurus, n = 20) cows were subjected to follicular aspiration without regard to the stage of their oestrous cycle (day of aspiration = D0) to remove all follicles ≥3 mm and induce growth of a new follicular wave. Ovaries were examined by ultrasonography on D4, D19, D34, D49 and D64, and antral follicles ≥3 mm were counted. Thereafter, cows were assigned to one of two groups: high or low antral follicular count (AFC, ≥30 and ≤15 antral follicles, respectively). After D64, ovaries were collected after slaughter and processed for histological evaluation. There was high repeatability in the numbers of antral follicles for all groups (range 0.77-0.96). The mean (±SD) numbers of antral follicles were 35 ± 9 (Bos indicus) and 38 ± 6 (Bos indicus-taurus) for the high AFC group and 10 ± 3 (Bos indicus) and 12 ± 2 (Bos indicus-taurus) follicles for the low AFC. The mean number of preantral follicles in the ovaries of Bos indicus-taurus cows with high AFC (116 226 ± 83 156 follicles) was greater (p < 0.05) than that of Bos indicus cows (63 032 ± 58 705 follicles) with high AFC. However, there was no significant correlation between numbers of antral and preantral follicles.

MALDI-MS lipid profiles of oocytes recovered by ovum pickup from Bos indicus and 1/2 indicus × taurus with high vs low oocyte yields.

The aim of the present study was to compare the lipid profile in oocytes of indicus and 1/2 indicus × taurus cows with high and low antral follicle count (AFC)/oocyte yields. After an OPU procedure (D0), antral follicles ≥3 mm were counted by ultrasonography (D4, 19, 34, 49, 64), and cows were assigned to groups with either high AFC (≥30 follicles; indicus, NH group; 1/2 indicus × taurus, AH group) or low AFC (≤15 antral follicles; indicus, NL group; 1/2 indicus × taurus, AL group). The lipid profiles of the oocytes were determined by MALDI-MS. For GI, GII and GIII oocytes, the indicus samples tend to cluster separately from the 1/2 indicus × taurus samples. The lipid species [PC (P-38:5) + H](+) and/or [PC (P-36:2) + Na](+) , [PC (38:2) + H](+) , [PC (38:5) + Na](+) and [TAG (60:8) + NH(4) ](+) were more abundant in indicus (NH and NL groups) than 1/2 indicus × taurus. The higher lipid content in the indicus oocytes likely reflects differences in the rate of lipid metabolism and may contribute to oocyte competence and embryo development.

Antral follicle populations and embryo production--in vitro and in vivo--of Bos indicus-taurus donors from weaning to yearling ages.

Interest in indicus-taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus-taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥ 3 mm as follows: G-High antral follicular count (AFC, n = 20, mean ≥ 40 follicles) and G-Low AFC (n = 20, mean ≤ 10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90-0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G-High vs G-Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre-pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus-taurus females.

Comparison of synthetic oviductal fluid and G1/G2 medium under low-1 oxygen atmosphere on embryo production and pregnancy rates in Nelore (Bos indicus) cattle.

In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low-oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low-oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.

Follicular dynamics, luteal characteristics, and progesterone concentrations in synchronized lactating Holstein cows with high and low antral follicle counts.

We performed two experiments in high milk-producing Holstein cows. First, we evaluated the repeatability of the antral follicle count (AFC) - where all follicles were ≥2 mm in diameter - during the days of the ovulation synchronization protocol for timed artificial insemination (TAI) in dairy cows (n = 20). In the second study we investigated the follicular dynamics and Doppler ultrasonography in cows with low (≤15 follicles, n = 9) and high (≥25 follicles, n = 9) AFC, and measured progesterone (P4) concentrations during CL evaluation. In addition, the diameter of the preovulatory follicle, area of blood flow of the preovulatory follicle, CL area, and CL blood flow were compared between groups with high and low AFC. Ultrasound examinations were performed during the days of the protocol and 10 days after ovulation to assess the CL. The constancy of the AFC was analyzed by calculating the repeatability. Parametric variables were analyzed using the t-test, and nonparametric variables were analyzed using the Mann-Whitney test (P ≤ 0.05). The repeatability of the AFC on different days of the protocol was 0.98. Cows with low AFC showed a greater diameter of the preovulatory follicle (16 ± 0.3 vs. 15 ± 0.4 mm), blood flow area of the preovulatory follicle (17.3 ± 2.3 vs. 8.1 ± 3.2 mm2), CL area (579.1 ± 16.9 vs. 405.8 ± 21.2 mm2), area of CL perfusion (97.1 ± 9.4 vs. 68.3 ± 5.2 mm2), as well as higher P4 levels (3.1 ± 0.2 vs. 2.2 ± 0.2 ng/mL). In high milk-producing Holstein cows, the AFC has high repeatability on different days of the TAI protocol, and cows with a low AFC show larger preovulatory follicles and CLs with greater perfusion, in addition to greater P4 concentrations in comparison to those cows with a high AFC.

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows.

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n=30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4+/-0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2+/-1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6+/-15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4+/-5.3) was higher (P<0.05) than the MOET method (6.7+/-3.7). However, pregnancy rates were lower (P<0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P>0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.

Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick-up.

This study evaluated the effects of the synchronization of ovarian follicular wave emergence on the efficiency of in vitro embryo production. Bos indicus cows (n = 20) were divided into two groups (control vs. synchronization) and subjected to repeated ovum pick-up (OPU) sessions (8 replicates each, with an interval of 21 days in a 2 × 2 crossover design) and subsequent in vitro embryo production. Cows in the control group (n = 10) were submitted to OPU procedures without any stimulation every 21 days. Animals in the synchronization group received a protocol-based progesterone implant, estradiol benzoate and prostaglandin on a random day of the estrus cycle (Day 0) and the OPU was performed on Day 5. After in vitro production, embryos were transferred to recipients synchronized at a fixed time and the diagnosis was performed 60 days later. An evaluation of the parameters for each OPU session revealed that donors that received the synchronization protocol pre-OPU showed a greater number of embryos (5.9 ± 0.5 vs. 4.5 ± 0.4; P = 0.037), higher rate of embryo production (45.8% vs. 38.5%; P = 0.001) and higher mean number of conceptions per group (2.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.07) in relation to the group that did not receive hormonal treatment. We concluded that synchronization of the follicular wave prior to OPU showed positive effects on in vitro embryo production as well as on pregnancy rates.

Alpha lipoic acid (ALA) effects on developmental competence of equine preantral follicles in short-term culture.

The effect of different concentrations of alpha lipoic acid (ALA) on the development and morphology of preantral follicles, as well as the proliferative activity of granulosa cells, was assessed after short-term culture. Ovaries (n = 5) of five seasonal anestrous mares were harvested in a local abattoir. At the laboratory, nine ovarian fragments (5 × 5 × 1 mm) from each animal were used. One fragment was immediately fixed in Bouin and subjected to histological and immunohistochemistry (proliferating cell nuclear antigen, PCNA) analyses (noncultured group; D0 = day 0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ plus ALA (50, 100, or 250 µM). After culture, fragments were subjected to histology and PCNA analyses. After two days of culture, ALA 50 and ALA 100 had the greatest (P < 0.05) percentage of normal primordial follicles (97.2 and 95.1%, respectively), when compared to other groups, and did not differ (P > 0.05) from the fresh noncultured control group. Furthermore, the total percentage of normal follicles was greater (P < 0.05) in the ALA 50 and ALA 100 than in the MEM-D2 group. After six days of culture, the highest (P < 0.05) proliferative activity of granulosa cells in developing follicles was observed for the groups MEM+ (92.9%), ALA 50 (100%), and ALA 100 (96.4%). In conclusion, the results of this study demonstrated that (1) ALA 50 and ALA 100 preserved the morphological integrity of equine primordial follicles for up two days of culture, and (2) granulosa cells of developing follicles enclosed in ovarian tissue and cultured for up to six days in MEM+ with or without ALA were highly stained by PCNA.

Correlation between phenotype, genotype and antral follicle population in beef heifers.

The present study was performed in indicus-taurus heifers 1) to determine if the antral follicle count (AFC) exhibits repeatability from puberty to yearling age and 2) to evaluate whether the phenotypic and genotypic parameters used in genetic improvement programs are correlated with AFC. In study I, Braford heifers (3/8 Nelore x 5/8 Hereford, n = 137) were serially examined by ultrasonography (with 60-day intervals) from weaning (9 ± 1 mo of age) to yearling ages (20 ± 1 mo of age) to monitor the numbers of antral follicles. In study II, the AFC of animals from experiment I and contemporary (same farm, considered at same age and kept under same conditions heifers n = 270 18-24 months) was correlated with the records of a genetic selection program using four statistical models with different covariates: i) model 1 considered effects of contemporary group and covariates age, weight gain from birth to weaning and visual scores for carcass traits at weaning, ii) model 2 covered contemporary group, age, weight gain from weaning to yearling and visual scores for carcass traits at yearling. The effects, variables and covariates of models 1 and 2 were combined to form model 3. Model 4 included the model 3 with addition of paternal effect. In study I, AFC varied from 3 to 64 follicles among females but was highly repeatable (0.89-0.92) within individuals in the same group. In study II, the four models tested showed low correlations with AFC: 0.072, 0.056, 0.082 and 0.172 for models 1, 2, 3 and 4, respectively. However, the model with paternal effect provided 17% of correlation of AFC and genotypic and phenotypic characteristics. Models 1, 3 and 4 also showed that AFC in indicus-taurus heifers can be influenced by finishing precocity at weaning (P < 0.05) with a variation of three follicles. Based on these studies, AFC in heifers from weaning to yearling age is highly variable between individuals and repeatable within the same female. Additionally, there is no correlation between phenotypic or genotypic characteristics and the antral follicle population. However, AFC can be slightly affected by finishing precocity at weaning.

Use of sexed sorted semen for fixed-time artificial insemination or fixed-time embryo transfer of in vitro-produced embryos in cattle.

Artificial insemination and in vitro embryo production are powerful tools for disseminating superior genetic qualities and improving the reproductive performance of dairy and beef cattle. In conjunction with these biotechnologies, sexed-sorted semen has been used to obtain offspring of a predetermined sex. This study compared the pregnancy rates obtained using in vitro fertilization/timed embryo transfer (IVF/TET) and timed artificial insemination (TAI), both performed using sexed-sorted (Y-chromosome-bearing) semen obtained from the same bull. For the in vitro embryo production, the ovaries of 250 Nelore cows with known histories were collected in the slaughterhouse and used for IVF. After evaluation of the recipients (IVF/TET group; n = 974), the resultant embryos were transferred to the females with corpus luteum (n = 822). The pregnancy-related data for this group were compared with those for the TAI group (n = 974). Ultrasonography was performed at 60 days to determine the pregnancy status and confirm the sex of the fetus. A total of 2008 oocytes produced 1050 embryos, with 52% of them reaching the blastocyst stage. The pregnancy rate and the accuracy in determining the fetal sex were 35.4% (345/974) and 95.07% (328/345), respectively, for the IVF/TET group and 30% (293/974; P < 0.05) and 94.88% (278/293), respectively, for the TAI group. In the present study, we concluded that male calves could be better obtained using IVF/TET rather than TAI; therefore, this strategy can be considered to increase the pregnancy rate of beef cattle.

Improvement in embryo recovery using double uterine flushing.

The objective of the present study was to evaluate the effects of double uterine flushing on the recovery of embryos/ova in cattle. Two hundred and ten embryo recovery procedures were conducted using a double uterine flushing method, and the results were compared with 432 conventional single-flushing procedures. Cyclic Limousin (n = 403) and Guzera (n = 239) donor cows received an intravaginal progesterone releasing device and 2 mg of estradiol benzoate on Day 0. Between Days 5 and 9, donors received decreasing doses of FSH, which ranged from 200 to 300 IU (Bos indicus) and 300 to 500 IU (Bos taurus). On the afternoon of Day 7, donors received an injection of 500 microg cloprostenol and progesterone implants were removed 12 h later (morning of Day 8). Artificial insemination was performed between 14 and 26 h after first detection of behavioral estrus. Cows were randomly assigned to have embryos recovered by a double-flushing method (n = 210) or the conventional single-flushing procedure (n = 432). For the double-flushing procedure, after first flushing the whole uterus with 1L of Dubelco's Phosphate Buffered Saline (DPBS), a Foley catheter was positioned in the uterine body to permit refilling of the uterus with fresh DPBS (80-150 mL). The catheter was closed with the plunger of a disposable 5 mL syringe, and the donors were allowed to rest in a holding area for 30 min. Thereafter, a second flush was performed to recover the solution remaining in the uterus. Animals from the control group were subjected to a single uterine flush. From 210 double-flushing procedures, 1409 viable embryos were recovered. In comparison, from 432 cows receiving the single-flushing procedure, 1993 embryos were recovered. Double flushing increased (P < 0.05) the number of embryos recovered per procedure compared to single flushing (6.7 +/- 0.4 versus 4.6 +/- 0.2, respectively; mean +/- S.E.M.). When double flushing was performed, average recovered embryos/ova increased (P < 0.05) from 8.3 +/- 0.4 to 12.7 +/- 0.7 in Limousin and from 7.9 to 11.5 in Guzera. Also, utilization of double flushing increased (P < 0.05) the number of viable embryos from 4.7 +/- 0.3 to 6.9 +/- 0.5 in Limousin and from 4.5 +/- 0.4 to 6.4 +/- 0.7 in Guzera. Mean total embryos/ova was similar (P > 0.05) between the control group and after the first uterine flushing in the double-flushing group; therefore, both flushings were conducted efficiently. In conclusion, double uterine flushing increased embryo recovery in cattle.

Population estimate of the preantral follicles and frequency of multioocyte follicles in prepubertal and adult bitches.

Oocytes from preantral follicles could be an alternative for in vitro maturation because most follicles are at the preantral stage. There are few studies that have sought to estimate the number of preantral follicles in bitches. Therefore, the aims of this study were to estimate the population of preantral follicles in the ovaries of small- and medium-sized prepubertal and adult bitches and compare the population of preantral follicles between the right and left ovaries and evaluate the frequency of multioocyte follicles (MOF). Eighty ovaries were collected by elective ovariohysterectomy from 40 healthy bitches. The bitches were divided into four groups: small-size prepubertal bitches (<10 kg, n = 20), medium-size prepubertal bitches (10-20 kg, n = 20), small-size adult bitches (<10 kg, n = 20), and medium-size adult bitches (10-20 kg, n = 20). Immediately after surgery, the ovaries were fixed in Bouin's solution and processed for histology. For each specimen, 70 histologic sections were cut and mounted on slides; then, the number of preantral follicles was estimated using a correction factor. The preantral follicles were classified according to the developmental stage. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for comparison between groups, and Fisher's exact test was used to evaluate the frequency of MOF (P ≤ 0.05). Considering the population of preantral follicles from the pair of ovaries, medium-size prepubertal bitches had the highest (P < 0.05) population of preantral follicles compared with the small and medium-size adult groups. There was a large variation in the numbers of preantral follicles among individuals of the same weight and within each group. There were differences between medium-size prepubertal and adult bitches regarding the population of preantral follicles in the right ovaries (145,482 ± 110,712 vs. 49,500 ± 44,821; P = 0.02); however, no differences were observed between the groups on the basis of comparisons of the number of preantral follicles in the left ovaries (P > 0.05). The prevalence of primordial MOF was higher in prepubertal bitches (47% vs. 28%), whereas adult bitches had a higher frequency of secondary MOF (49% vs. 25%; P < 0.05). We conclude that medium-size prepubertal bitches had the highest population of preantral follicles compared with small and medium-size adult bitches, and the use of only one ovary per bitch implied contrasting result. The presence of primordial MOF was higher in prepubertal bitches and at the secondary stage in adult bitches.

Improvement of development of equine preantral follicles after 6 days of in vitro culture with ascorbic acid supplementation.

The aim of this study was to evaluate the effects of different concentrations of ascorbic acid (25, 50, and 100 µg/mL) in supplemented minimum essential medium (MEM+) on the development of equine preantral follicles that were cultured in vitro for 2 or 6 days. The contralateral ovaries (n = 5) from five mares in seasonal anestrus were collected from a local abattoir. Nine ovarian tissue fragments of approximately 5 × 5 × 1 mm were obtained from each animal. One fragment was immediately fixed and subjected to histologic analysis (control group; Day 0), and the other eight were placed in PBS supplemented with penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4 °C for 1 hour (during transport to the laboratory). The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ plus ascorbic acid at three different concentrations, establishing the following nine groups: control; MEM+ (D2); MEM+ (D6); MEM+ 25 µg/mL of ascorbic acid (D2); MEM+ 25 µg/mL of ascorbic acid (D6); MEM+ 50 µg/mL of ascorbic acid (D2); MEM+ 50 µg/mL of ascorbic acid (D6); MEM+ 100 µg/mL of ascorbic acid (D2); and MEM+ 100 µg/mL of ascorbic acid (D6). The preantral follicles were classified according to their stage (primordial, primary, secondary, or antral) and their morphology (normal or abnormal). Slides (n = 951) including 4450 histologic sections were evaluated. Follicles were observed in only 4.85% (216 of 4450) of the histologic sections. Of the 407 follicles evaluated, 120 were in the primordial stage and 287 were in different developmental stages; additionally, 43.5% were morphologically normal. After 6 days of culture, the groups cultured with 50 and 100 µg/mL of ascorbic acid differed in terms of follicular development compared with the other groups. On the basis of occurrence of follicular development and the presence of viable follicles, it can be concluded that a positive effect of culture for 6 days in MEM+ supplemented with 50 and 100 µg/mL of ascorbic acid was observed on equine ovarian fragments.

Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery.

We evaluated the relationship between follicle size and oocyte recovery (OR) using ultrasound-guided follicle aspiration. Thirty Holstein cows were subjected to OR without gonadotrophic therapy. Oocytes were recovered two to four times from each cow in a total of 67 aspiration sessions. Ovarian follicles with diameters < or =4 mm and >4 mm were aspirated in separated groups. Recovered oocytes from each group were kept separate and submitted to in vitro maturation, fertilization, and culture to the blastocyst stage. A total of 430 follicles were aspirated, of which 154 (35.8%) were from follicles >4 mm and 276 (64.2%) were from follicles < or =4 mm. Seventy-seven oocytes (50%) were recovered from follicles >4 mm and 200 (72.2%) were from follicles < or =4 mm. Nineteen blastocysts were obtained from follicles >4 mm, whereas 45 blastocysts were obtained from follicles < or =4 mm. Recovery rate was greater (P<0.01) in follicles < or =4 mm. Oocyte quality, cleavage rate and blastocyst development did not differ between different follicle sizes. Routine aspiration of small follicles (< or =4 mm) could increase the number of oocytes available for in vitro development.

Efficacy of linear and convex transducers for ultrasound-guided transvaginal follicle aspiration.

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n=28) and convex (n=38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H(2)O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P<0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle.