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Contents: 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. [ABSTRACT FROM AUTHOR]

Bos taurus indicusfemales provide a greater in vivooocyte recovery (2-4 times more) in comparision with Bos taurus taurus. This aspect has strongly contributed for the success of the embryo industry in Brazil. Little information is available to explain this difference. Our objective was to test the hypothesis that the difference in oocyte yield is a result of higher numbers of preantral follicles in the ovaries of B. indicusfemales. Ovaries (n= 44) of fetuses aging from 180 to 240 days and of heifers from 20 and 24 months were collected from Bos taurus indicus(Nelore) and Bos taurus taurus(Aberdeen Angus) females at a slaughterhouse. Fetal age was estimated from the crown-rump length (Evans HE and Sack WO 1973 Anat. Hist. Embryol. 2, 11-45). Ovaries from Nelore fetuses (n= 10) and heifers (n= 12) and Aberdeen Angus fetuses (n= 10) and heifers (n= 12) were cut longitudinally into two halves, fixed in Bouin''s solution, and processed for histological evaluation. The ovarian halves were dehydrated in alcohol, cleared with xylene, embbebed in paraffin, and serially sectioned at 7 μm. Every 120th section was mounted and stained with periodic acid Schiff and hematoxylin. The number of preantral follicles was estimated through the counting of follicles in each section using the nucleus of the oocyte as a marker and a correction factor (Gougeon A and Chainy GBN 1987 J. Reprod. Fertil. 81, 433-442). Only 1 ovary per female was analyzed. Preantral follicles were classified according to the developmental stage as primordial (1 layer of flattened granulosa cells surrounding the oocyte), primary (1 layer of cuboidal granulosa cells), or secondary (2 or more layers of cuboidal granulosa cells), and as normal or degenerated according to their morphological appearance. The number of preantral follicles was not normally distributed and was compared using the Mann-Whitney test. There was no difference (P> 0.05) between the average number of preantral follicles in the ovaries of Bos indicusand Bos taurusfemales. The average number of preantral follicles per female was 143 929 ± 253 (mean ± SD) and 285 155 ± 570 for Bos taurusand Bos indicusfetuses, respectively, and 76 851 ± 280 and 109 673 ± 293 for Bos taurus andBos indicus heifers (P> 0.05), respectively. A large variation in numbers of preantral follicles was observed among individuals within the same category and between breeds. Our results suggest that there might be differences in mechanisms controlling follicle development after the preantral stage accounting for the greater oocyte yield from Bos indicusfemales. This work was supported by grants from the Universidad Nacional del Litoral (CAI+D program), Universidad Nacional de Lomas de Zamora, the Argentine National Agency for the Promotion of Science and Technology (ANPCyT), and the Argentine National Council for Science and Technology (CONICET). [ABSTRACT FROM AUTHOR]

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey''s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P< 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P< 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P< 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1of a-tocopherol after 6 d. After 8 days of in vitroculture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitroculture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships. [ABSTRACT FROM AUTHOR]

The objective of experiment 1 was to evaluate the influence of follicular wave moment on the quality and number of recovered oocytes through ovum pick-up (OPU), as well as blastocyst rate, in Nelore cows. Two hypotheses were tested: (1) the number of recovered viable COC would be higher at Days 3 and 5 than at Days 7 and 9; (2) the COC aspirated at the beginning of the follicular wave (Day 3 or Day 5) would be more competent and would produce more blastocysts than COC obtained at the end of the wave (Day 7 and Day 9), because these would be in a certain degree of advanced atresia. Cows (n= 10) received 2 mL of estradiol benzoate (Estrogin®), an auricular progestagen implant (Crestar®) for 7 days, and 2 mL of PGF2 analog (Preloban®) at the moment of implant retrieval. After 24 h animals were submitted to aspiration of the dominant follicle (ADF), this moment being considered Day 0. After ADF the donors were submitted to OPU on Days 3, 5, 7, and 9 (groups D3, D5, D7, and D9, respectively) of the new follicular wave. Each animal was submitted to all treatments. In each phase the protocol began with a 2-day interval from the group D9, D7, D5, and D3. Recovered COC went to IVM, IVF, and IVC in the same session of IVP. Statistical analysis were performed with SAS software (SAS Institute, Cary, NC, USA), using ANOVA at a 5% level of significance. There was no difference in the total number of follicles visualized at the moment of OPU among groups (D3:195, D5:189, D7:161, D9:151) or in the recovery rate (77.94; 78.30; 72.04; 73.50) and number of recovered total structures (152, 148, 116, 111). There was no difference in the total number of viable COC (133, 137, 103, 97) or in cleavage rate of COC in the 4 groups (79.69; 83.45; 71.84; 76.28). The total number and the blastocyst rates were higher in groups D3 and D5 (66, 49%, and 62, 45.25%) than in groups D7 and D9 (34, 33%, and 35, 36.08%). Statistical analysis was performed with the chi-square test, with a significance level of 5%. In experiment 2 nuclear and cytoplasmatic maturation were evaluated in COC recovered at Days 4 and 8 regarding nuclear maturation (progression to metaphase II stage) and cortical granule migration, respectively. The hypothesis tested was that COCs recovered at the beginning of the follicular wave would have a higher competence and therefore higher maturation rate compared with those the aspirated at the end. There was no difference in the number of viable COC (78 v.97), percentages of nuclear maturation (74.35 v.53.6), and cytoplasmatic maturation (48.71 v.30.92). The results of the 2 experiments demonstrated that the beginning wave (Days 3 to 5) is the most appropriate moment for obtaining oocytes destined for IVP because of the greater oocyte competence and consequently, larger number and increased blastocyst production rates. Moreover, the results obtained in experiment 2 indicate that the low rate of cytoplasmic maturation can be a major limiting factor of IVP in Nelore cows. FAPESP. [ABSTRACT FROM AUTHOR]

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitroculture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin''s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey''s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P≤ 0.05) and the percentage of growing follicles was increased (P≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitrofor 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship. [ABSTRACT FROM AUTHOR]

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro(Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicusanimals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicusis higher than those for European breeds (Pontes JHF et al.2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n= 90,086) and viable (n= 64,826) oocytes and the number of embryos produced in vitrofrom Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n= 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n= 64,826) were in vitromatured for 24 h at 38.8°C in an atmosphere of 5% CO2in air. Since this was a commercial programm, frozen sexed semen (2 × 106mL-1) from Gir (n= 8) or Holstein (n= 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n= 617), 11.4 ± 3.9 for Holstein cows (n= 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n= 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n= 37, P< 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P< 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n= 12,243/3378) for Gir cows, 2.2 (n= 2426/1138) for Holstein cows, 3.9 (n= 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n= 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicusdonor cows was greater (P< 0.01) than from Bos indicuscows. The number of recoverable and viable oocytes and the number of embryos produced in vitrofrom Bos indicusdonors were higher than from Bos taurusfemales. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicusfemales. This work was supported by In Vitro Brasil. [ABSTRACT FROM AUTHOR]

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 mm 2 ), CL area (579.1 ± 16.9 vs. 405.8 ± 21.2 mm 2 ), area of CL perfusion (97.1 ± 9.4 vs. 68.3 ± 5.2 mm 2 ), 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2017 Blackwell Verlag GmbH.)

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., (Copyright © 2016. Published by Elsevier Inc.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2015 Blackwell Verlag GmbH.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© 2014 Blackwell Verlag GmbH.)

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., (© 2014 Blackwell Verlag GmbH.)

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2013 Blackwell Verlag GmbH.)

The aim of this study was to compare four methods of estrus resynchronization performed 23 days after timed artificial insemination (TAI) plus estrus observation in Bos indicus cows. Eight hundred fourteen lactating Nelore cows were submitted to TAI and then randomly assigned to one of the five following treatments: R23 (resynchronization without eCG), R23/200 (resynchronization with 200 IU of eCG), R23/300 (resynchronization with 300 IU of eCG), R23/TCR (resynchronization with temporary calf removal [TCR]), and a control group, with estrus observation followed by AI (with no resynchronization). Treatment consisted of a progesterone device plus administration of estradiol benzoate on Day 0; on Day 8, the device was removed and cloprostenol was applied, together with estradiol cypionate. Also on Day 8, either eCG was administered or TCR was performed in the resynchronized groups, except for R23. The females were inseminated 48 hours after device removal or TCR (33 days after the first TAI). The control group was kept under estrus observation from 18 to 23 days after the first TAI and was inseminated 12 hours after detection of estrus. The first pregnancy evaluation was performed using ultrasound examination 31 days after the first TAI. After 30 days of the resynchronization, a second pregnancy evaluation was performed and the animals in the R23/300 and R23/TCR groups achieved the highest conception rates, 76.6% and 74.0%, respectively (P < 0.05). There were no differences between the conception rates of the animals in the R23/200 (63.3%), R23 (61.3%), and control (54.3%) groups (P > 0.05). These results suggest that estrus resynchronization at 23 days after TAI can effectively improve the conception rate of lactating Bos indicus cows in a short time period. Furthermore, resynchronization with 300 IU of eCG or with TCR provided the best results., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2012 Blackwell Verlag GmbH.)

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up., (Copyright © 2011 Elsevier Inc. All rights reserved.)