Relationships between antral follicle count and reproductive characteristics of embryo-recipient mares.

Mares (n = 77) were evaluated by antral follicle count (AFC) and selected as embryo recipients. Cyclic recipients received embryos between days 4-6 after ovulation. The acyclic recipients received an intramuscular (i.m.) protocol with 5mg of estradiol benzoate (EB) on the day of donor ovulation (D0; D-4 recipient), 3mg of EB on the following day (D1; D-3 recipient), and 3mg of EB (D2; D-2 recipient). Furthermore, 1500mg of progesterone (P4) i.m. given on D0 of the recipient (D4 donor) followed by 1500mg of P4 on the day of ET (D4-6 recipient). On the ET day, the AFC and animals' weight, body condition score (BCS), corpus luteum diameter, age and degree of uterine edema (UE) were measured. Pregnancy was confirmed on days 12 and 30. Low AFC was defined as ≤11 follicles (n = 43 mares) and high AFC as >11 follicles (n = 34 mares). Data were analyzed by a mixed effect model, including AFC group, reproductive seasonality, and season (P ≤ 0.05). UE was influenced (P = 0.05) by reproductive seasonality. The conception rate was higher (P = 0.016) in recipients with low (79.07 %) than high AFC (61.76 %) and higher (P = 0.005) in cyclic (81.40 %) than anestrus (58.82 %) mares. In addition, we observed a tendency (P = 0.06) for the interaction of AFC*reproductive seasonality, showing that high*anoestrus recipients had the lowest conception rate (37.50 %b) compared to high*cyclic (83.33 %a), low*anoestrus (77.78 %a) and low*cyclic (80 %a). The conception rate was higher in cyclic recipients with low AFC. Furthermore, UE was influenced by reproductive seasonality and mares in anestrus showed a higher degree of UE than cyclic mares.

Effects of maternal antral follicle count in Bos taurus indicus cattle on the genetic merit of male offspring and antral follicle count of female offspring.

This study evaluated the effects of antral follicle count (AFC) in female cattle on offspring characteristics. Recently calved multiparous Bos indicus cows (Nelore; n = 222) were evaluated using ultrasonography on random days of their estrous cycle to determine the AFC and were classified into "low" (≤15 follicles), "intermediate" (≥16 and ≤ 29 follicles), and "high" (≥30 follicles) AFC groups. Weight and scrotal circumference (SC) of male offspring from these cows (n = 127) were determined from 20 to 27 months, and the data were added to a genetic evaluation program (economic total genetic merit, MGTe and TOP value) that uses the kinship matrix to evaluate the genetic relationship between animals. The AFC of female offspring from these cows (n = 95) was evaluated to analyze the relationship between the AFC of mothers and daughters. The effects of maternal AFC on the genetic merit of male and female offspring were analyzed using GLIMMIX and GLM, respectively. Correlations were assessed using the Pearson's coefficient. Male offspring of cows with high AFC had superior MGTe (P = 0.005) and TOP values (P = 0.01) than those from cows with low AFC. Additionally, the AFC of mothers was positively correlated with MGTe (R = 0.33; P < 0.0001) and negatively correlated with TOP values (R = -0.32; P < 0.0001). The SC (P = 0.01), but not body weight of the offspring (P = 0.46) was affected by maternal AFC. The daughters' AFC were correlated (R = 0.29; P = 0.004) with mothers' AFC and were influenced by maternal (P = 0.05) but not paternal (P = 0.77) effect. In conclusion, cows with high AFC produced males with greater MGTe, superior TOP values and higher SC. Maternal AFC did not influence the weight of male offspring but was correlated with the AFC of daughters.

DNA methylation associated with polycystic ovary syndrome: a systematic review.

PURPOSE: Polycystic ovary syndrome (PCOS) is an endocrine metabolic disease that affects women of reproductive age and is one of the main causes of anovulatory infertility. However, the cause of PCOS is yet fully understood, and genetic factors play an important role in its etiology. In this study, we reviewed the main genes involved in the etiology of PCOS and the influence of DNA methylation, aiming to answer the study´s guiding question: 'What is the influence of DNA methylation on the main genes involved in PCOS?'. METHODS: We used the MEDLINE database, and inclusion criteria (primary and original articles, written in English, found through our entry terms) and exclusion criteria (literature reviews and articles that used animals to perform the experiments and that focused in other epigenetics mechanism without being DNA methylation) were applied. RESULTS: Twenty-three scientific articles, from a total of 43 articles read in full, were chosen for this study. Eighteen studies confirmed DNA methylation associated with PCOS. CONCLUSION: The most relevant genes related to PCOS were INSR, LHCGR, and RAB5B, which may be epigenetically altered in DNA, with the first two genes hypomethylated and the last hypermethylated. The epigenetic changes presented in the genes related to PCOS or their promoters were only at the CpG sites.

Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers.

The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.

Progesterone Device Use Improves Ovum Pick-Up Efficiency in Acyclic Donors.

The aim of this study was to evaluate follicular dynamics and ovum pick-up (OPU) efficacy in untreated mares or mares treated with an intravaginal progesterone (P4) device during seasonal anestrus (acyclic) and during the breeding season (cyclic). Six mares (mean age = 5 years), were recruited into an ovum pick-up scheme that was performed every 14 days with and without the P4 device, during the acyclic and cyclic phases. Aspirations amounted to seven procedures with or without the P4 device during each phase. Five ultrasound assessments were performed at each interval between the OPUs. Data on follicular number and diameter as well as the numbers of recovered and the percentage of recovered oocytes were also collected. The number of follicles from mares in the acyclic phase was higher (P < .005) regardless of the treatment. However, the follicular diameter was smaller for the P4 group (P < .005) from the 2nd to the 5th evaluation post-OPU procedure. The percentage of oocytes recovered during the acyclic phase was higher for mares treated with the P4 device (P < .005). The P4 device resulted in follicles with smaller diameters and facilitated OPU efficacy.

Conception rate and pregnancy loss in fixed-time cattle embryo transfer programs are related to the luteal blood perfusion but not to the corpus luteum size.

This study aimed to evaluate the effect of luteal blood perfusion and corpus luteum (CL) area on the conception rate and occurrence of pregnancy loss of recipients in a large-scale fixed-time embryo transfer (FTET) program. Multiparous Brangus cows (n = 1700) at 45 days postpartum and body condition scores (BCS) between 2.5 and 4.0 (3.0 ± 0.3) were used in this study. On a random day of the estrous cycle (day -10), the females received progesterone and estradiol based on the FTET protocol. On day 7, 1465 recipients had at least one CL and were evaluated using B-mode ultrasound for the CL area (cm2) and color Doppler for the luteal blood perfusion score (I/low-vascularization area <40% of the CL; II/medium-vascularization >45% to < 50%; and III/high-vascularization >50%). Immediately after CL evaluation, each recipient received a single fresh embryo (blastocyst stage) ipsilateral to the CL, in vitro produced from a commercial laboratory. Pregnancy diagnosis was performed at 30 days and repeated 60 days later to evaluate pregnancy loss (30-90 days). Ultrasound evaluation and embryo transfer were performed by a single technician. For data analysis, in addition to luteal blood perfusion groups, recipients were retrospectively ranked according to CL area into small (<3 cm2; 2.63 ± 0.01), medium (>3 to < 4 cm2; 3.44 ± 0.01), and large (>4 cm2; 4.77 ± 0.03). Data were analyzed using a logistic regression model (P < 0.05). The overall conception rate was 44.2% (648/1465), influenced by the luteal blood perfusion score [P = 0.03; high 48.4%a (134/277), medium 44.6%a (427/958), and low 37.8%b (87/230)] but not by CL area ranking [P = 0.37; large 41.8% (225/538), medium 45.2% (276/610), and small 46.4% (147/317)]. There was no interaction between the luteal blood perfusion score and CL area ranking (P = 0.81), and the BCS did not affect the results of this study (P = 0.51). In terms of pregnancy loss up to 90 days, there was no effect on the CL area ranking (P = 0.77), but the flow score showed an effect [P = 0.03; high 3.6%b (5/139), medium 9.3%a (44/471), and low 10.3%a (10/97)]. The conception rate and occurrence of pregnancy loss in the FTET program in beef cattle are related to luteal blood perfusion but not CL size.

Investigation of the vaginal microbiota of dairy cows through genetic sequencing of short (Illumina) and long (PacBio) reads and associations with gestational status.

The vaginal microbiota has been shown to be important in local immune regulation and may play a role in reproduction and fertility. Next-generation sequencing (NGS) technologies have been used to characterize the bovine vaginal microbiota, mainly using short-read sequencing (Illumina). However, the main limitation of this technique is its inability to classify bacteria at the species level. The objective of this study was to characterize the bovine vaginal microbiota at the species level using long-read sequencing (PacBio) and to compare it with the results of short-read sequencing. In addition, the vaginal microbiota of cows that became pregnant after artificial insemination (AI) was compared with that of infertile animals. Thirteen Holstein cows had vaginal swabs collected prior to AI. DNA was extracted and subjected to Illumina and PacBio sequencing to characterize the V4 region and the entire 16S rRNA gene, respectively. PacBio sequencing yielded 366,509 reads that were assigned to 476 species from 27 phyla. However, none of the most abundant reads (>1%) could be classified at the species level. Illumina sequencing yielded more reads and consequently was able to detect a more observed species, but PacBio sequencing was able to detect more unique and rare species. The composition of the vaginal microbiota varies according to the sequencing method used, which might complicate the interpretation of results obtained in the majority of the current studies. The present study expands on the current knowledge of bovine microbiota, highlighting the need for further efforts to improve the current databanks.

Spatial distribution of preantral follicles in ovarian parenchyma of bovine species.

The goal of this study was to determine the distribution of preantral follicles in bovine ovaries. Follicular distribution in the ovaries (n = 12) was evaluated in the region of the greater curvature of the ovary (GCO) and the region close to the ovarian pedicle (OP) of Bos taurus indicus heifers of the Nelore breed. Two fragments were obtained from each region of the ovary (GCO and OP). The mean weight of the ovaries was 4.04 ± 0.32 g. The mean antral follicle count (AFC) was 54.58 ± 3.55 follicles (minimum and maximum variation of 30 and 71 follicles, respectively). In total, 1123 follicles were visualized in the region of the GCO; 949 (84.5%) of them were primordial follicles and 174 (15.5%) were developing follicles. The region close to the OP contained 1454 follicles, of which 1266 (87%) were primordial follicles and 44 (12.9%) were developing follicles. The OP region showed a higher proportion of intact follicles in the primordial (P < 0.0001) and primary (P = 0.042) stages compared with the GCO region. The proportion of secondary follicles was similar in the OP and GCO regions. The ovaries of two bovine females (16%; 2/12) contained multi-oocytes follicles, which were characterized as primary follicles. Therefore, the distribution of preantral follicles in the bovine ovary was heterogeneous, with the region close to the OP containing a greater number of preantral follicles compared with the GCO region (P < 0.05).

Evaluation of hCG as gonadotropic support to timed embryo transfer protocol in beef cattle.

This study compared the reproductive performance of embryo recipients treated with a timed embryo transfer (TET) protocol using human chorionic gonadotropin (hCG) or equine chorionic gonadotropin (eCG). On a random day of the estrous cycle (Day -10) indicus-taurus recipients (n = 341; 194 nulliparous and 147 multiparous cows) with a body condition score between 3.0 and 4.0, were submitted to the TET protocol consisting of an intramuscular (i.m.) injection of 2.0 mg estradiol benzoate (EB) and the insertion of intravaginal progesterone (P4) device that remained until Day -2.5. On the same day (-2.5), the recipients received i.m. 150 mg D-cloprostenol and 1 mg estradiol cypionate and were randomly divided into two groups: the eCG group (n = 179), in which females received i.m. 300 IU eCG and the hCG group (n = 162), in which females received 150 IU hCG. Then, estrus intensity and the diameter of the dominant follicle (DF) were monitored on D0 and the quality of the corpus luteum (CL) (B mode and color Doppler) was assessed on D7 to select recipients eligible for receiving the transfer of an embryo produced in vitro. Pregnancy diagnosis was assessed 23 days after the transfer. Continuous data were analyzed by ANOVA using a mixed-effects model and Tukey's test. The rates were analyzed using a logistic regression model. The diameter of the DF on day 0 of the TET protocol was influenced by the interaction between gonadotropic treatment and category (P = 0.01), and nulliparous recipients treated with hCG had the smallest diameter. Treatment with hCG and eCG resulted in a high rate of estrus expression; however, the proportion of females with a high-intensity of estrus was higher in the hCG group (79.84 vs. 68.61%, respectively; P = 0.03). The utilization rate (recipients with CL) showed a tendency (P = 0.06) to be influenced by the interaction between gonadotropic treatment and category, wherein nulliparous recipients treated with hCG exhibited a lower utilization rate than the other groups. The diameter, perimeter, and area of the CL were similar (P > 0.1) in all groups. However, the hCG group resulted in CL with a better Doppler evaluation score (P = 0.04), central blood flow (P = 0.03), and tendency towards greater peripheral blood flow (P = 0.08). The rates of conception (32.00% hCG vs. 35.10% eCG; P = 0.46) and pregnancy (24.69% hCG vs. 29.61% eCG; P = 0.20) were similar between the hCG and eCG groups. However, an interaction between the gonadotropic treatment and category revealed lower conception (P = 0.01) and pregnancy rates (P = 0.001) in nulliparous recipients treated with hCG. Treatment with hCG resulted in a greater intensity of estrus expression and CL with a higher Doppler score, which determined rates of utilization, conception, and pregnancy similar to conventional protocols using eCG. However, nulliparous recipients treated with hCG exhibited a lower overall reproductive rate.

Manipulação de oócitos inclusos em folículos ovarianos pré-antrais e a importância dos antioxidantes ­ revisão de literatura / Manipulation of oocytes included in preantral ovarian follicles and the importance of antioxidants ­ literature review / Manipulación de ovocitos incluidos en folículos ováricos preantrales e importancia de los antioxidantes - revisión de la literatura

A manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA) vem sendo estudada pensando na perspectiva futura de aplicação direta na reprodução humana, principalmente para mulheres que sofrem de doenças ou que precisam passar por tratamentos que interferem na função ovariana. Nesse contexto, o objetivo deste trabalho é revisar aspectos relacionados com a biotécnica de MOIFOPA e a importância dos antioxidantes no cultivo in vitro de folículos pré-antrais. Foi realizada uma pesquisa na base de dados PubMed, buscando artigos sobre a biotécnica, principalmente relacionados com a necessidade do uso de antioxidantes no cultivo. A grande maioria dos estudos sobre a biotécnica utilizam como modelo experimental os folículos ovarianos de diferentes espécies de animais. A MOIFOPA compreende o isolamento e o resgate de folículos ovarianos pré-antrais provenientes de ovários, seguido da conservação através da técnica de resfriamento ou congelação e o cultivo folicular in vitro, a fim de promover o crescimento, a maturação e a fecundação in vitro (FIV) dos oócitos inclusos nesses folículos, maximizando o potencial reprodutivo feminino e diminuindo a atresia folicular que acontece in vivo. Um aspecto que pode interferir no sucesso do cultivo in vitro de folículos ovarianos pré-antrais é a produção em excesso de espécies reativas de oxigênio (EROs). Os ácidos ascórbico e alfa lipóico vem demonstrando resultados interessantes para reduzir os efeitos que as EROs causam sobre os folículos ovarianos pré-antrais cultivados in vitro.

The manipulation of oocytes included in preantral ovarian follicles (MOEPF) has been studied considering the future perspective of direct application in human reproduction, especially for women who suffer from diseases or who need to undergo treatments that interfere with ovarian function. In this context, the objective of this paper is to review aspects related to the biotechnology of MOIFOPA and the importance of antioxidants. A search was carried out in the PubMed database, searching for articles on biotechnology, mainly related to the need to use antioxidants in cultivation. The vast majority of studies on biotechnology use ovarian follicles from different species of animals as an experimental model. MOIFOPA comprises the isolation and rescue of preantral ovarian follicles from ovaries, followed by conservation through the cooling or freezing technique and in vitro follicular cultivation, in order to promote growth, maturation and in vitro fertilization ( IVF) of the oocytes included in these follicles, maximizing the female reproductive potential and decreasing the follicular atresia that occurs in vivo. One aspect that may interfere with the success of in vitro culture of preantral ovarian follicles is the excess production of reactive oxygen species (ROS). Ascorbic and alpha lipoic acids have shown interesting results in reducing the effects that ROS cause on in vitro cultured preantral ovarian follicles.

manipulación de ovocitos incluidos en folículos ováricos preantrales (MOIFOPA) se ha estudiado con la perspectiva futura de su aplicación directa en la reproducción humana, especialmente en mujeres que padecen enfermedades o que necesitan someterse a tratamientos que interfieren en la función ovárica. En este contexto, el objetivo de este trabajo es revisar los aspectos relacionados con la biotécnica de MOIFOPA y la importancia de los antioxidantes en el cultivo in vitro de los folículos pré-antrais. Se realizó una investigación en la base de datos PubMed, buscando artículos sobre la biotecnología, principalmente relacionados con la necesidad del uso de antioxidantes en el cultivo. La mayoría de los estudios sobre biotecnología utilizan como modelo experimental los folículos ováricos de diferentes especies de animales. El MOIFOPA incluye el aislamiento y rescate de los folículos ováricos preantrales de los ovarios, seguido de su conservación mediante la técnica de enfriamiento o congelación y el cultivo folicular in vitro, con el fin de promover el crecimiento, la maduración y la fecundación in vitro (FIV) de los ovocitos incluidos en estos folículos, maximizando el potencial reproductivo femenino y disminuyendo la atresia folicular que se produce in vivo. Un aspecto que puede interferir en el éxito del cultivo in vitro de folículos ováricos preantrales es la producción excesiva de especies reactivas de oxígeno (ROS). El ácido ascórbico y el ácido alfa lipoico han mostrado resultados interesantes para reducir los efectos que causan las ERO en los folículos ováricos preantrales cultivados in vitro.

Evaluation of three classification methods of antral follicle count and fertility to the timed artificial insemination in cattle.

The controversial data about antral follicle count (AFC) may be partially explained by the different criteria used to determine what is high, intermediate and low AFC. This study evaluated different classification methods for AFC groups, relating them to the conception rate, dominant follicle size and body condition score (BCS) in cows submitted to timed artificial insemination (TAI). Nelore cows (Bos indicus; n = 935), received a reproductive program consisting of TAI and natural breeding. Conception rate, BCS and dominant follicle size during TAI were evaluated by three AFC methodologies: i) mean and standard deviation: low (≤ 15 follicles); intermediate (≥ 16 to ≤ 44 follicles) or high (≥ 45 follicles); ii) quartiles: low (≤ 15 follicles); intermediate (≥ 16 to ≤ 39 follicles), or high (≥ 40 follicles); and iii) AFC score: I (low; ≤ 15 follicles); II (intermediate; ≥ 16 to ≤ 30 follicles); III (high; ≥ 31 to ≤ 44 follicles) or IV (very high; ≥ 45 follicles). Data were analyzed by a GLIMMIX and Tukey test or binary logistic regression model (P ≤ 0.05). The conception rate to TAI was influenced (P < 0.05) by AFC in the three methods classification, being the highest conception rate observed in the low AFC group regardless of method utilized: Mean (low 61.73%a, intermediate 54.02%ab and high 49.48%b), Quartiles (low 61.73%a, intermediate 53.59%ab and 51.46%b) and Score (I 61.73%a, II 54.80%ab, III 53.23%ab and IV 49.48%b). There were variations (P < 0.05) in the conception rate within the 2.50 to 2.75 BCS range for all AFC classification methods, with the low AFC females presenting the best results, regardless of the method used. Also, females with low AFC showed larger (P < 0.05) diameters of dominant follicles at the TAI regardless of method. The different methodologies used (Mean, Quartile and Score) to AFC classification showed a consistency between the main findings, and we believe that this standardization will facilitate the interpretation of data involving AFC.

Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid.

The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher's exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

Culture of preantral ovarian follicles of Bos taurus indicus with alpha-lipoic acid.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at -80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

Impact of the antioxidant quercetin on morphological integrity and follicular development in the in vitro culture of Bos indicus female ovarian fragments.

We evaluated the effect of quercetin on the in vitro culture of bovine ovarian fragments in relation to morphology, development, and oxidative stress. Ovaries (n = 12) from Nelore heifers (n = 6) were used. Each pair of ovaries was divided into nine fragments, and one fragment from each animal was fixed in Bouin solution for 24 h (histology control) or frozen (- 80°C; control for oxidative stress). Other ovarian fragments (n = 8) were distributed into concentrations of 0, 10, 25, and 50 µg/mL of quercetin added to the culture medium for 5 or 10 d. Data were analyzed by chi-square test or ANOVA followed by Tukey's test (P < 0.05). Treatment with 25 µg/mL quercetin resulted in the highest proportion of total intact follicles for 5 (67.3%) and 10 d (57.1%); the concentration of 25 µg/mL also presented the best proportion of developing follicles for 5 d (68.7%) and 10 d (62.8%). Treatment with 25 µg/mL quercetin resulted in significant ferric reduction for 10 d of culture, but not for 5 d. No difference (P > 0.1) was observed in the production of reactive oxygen species or in the oxidative degradation of lipids between treatments and non-cultivated controls. Treatment with 25 µg/mL quercetin preserved the morphological integrity of the developing follicles for 5 and 10 d of culture, in addition to promoting the best antioxidant potential after 10 d of culture in bovine ovarian fragments.

Retirada de corpo estranho (dispositivo impregnado com progesterona) da cavidade abdominal de novilha da raça Jersey ­ relato de caso / Removal of a foreign body (progesterone impregnated device) from the abdominal cavity of a Jersey heifer ­ a case report / Extracción de cuerpo extraño (dispositivo impregnado con progesterona) de la cavidad abdominal de novilla raza Jersey - reporte de caso

A inseminação artificial em tempo fixo (IATF) é uma biotécnica com inúmeras vantagens para a produção de bovinos. Para a realização da IATF, as fêmeas bovinas devem receber um protocolo farmacológico que induz a ovulação de forma sincronizada. Esse protocolo inicia com a inserção de um dispositivo impregnado com progesterona que deve permanecer no fundo vaginal da fêmea por tempo determinado. O presente relato tem por objetivo descrever a remoção de um implante impregnado com progesterona que foi encontrado na cavidade abdominal de uma novilha. Durante um atendimento reprodutivo de rotina em uma propriedade rural, uma novilha da raça Jersey, recém-adquirida, com histórico de ter sido submetida à IATF, foi avaliada para diagnóstico de gestação. No exame a novilha não estava gestante e apresentava um cisto folicular ovariano. A novilha foi submetida novamente à IATF em que foi inserido e removido um implante, com isso o cisto regrediu e ao inseminar a novilha não foi possível passar a cérvix com o aplicador. Vinte dias após a inseminação a novilha manifestou estro e ao ser avaliada para nova inseminação, foi identificado um implante solto na cavidade abdominal da novilha. Foi realizada uma laparotomia pelo flanco esquerdo e o implante foi removido. Após a recuperação da cirurgia a novilha foi inseminada e novamente e ficou gestante. Os protocolos de IATF devem ser realizados por profissionais capacitados para evitar problemas como a presente situação em que um implante foi encontrado na cavidade abdominal de uma novilha.(AU)

The fixed-time artificial insemination (FTAI) is a biotechnique with numerous advantages for cattle production. In order to perform the FTAI, the cows must receive a pharmacological protocol to induce ovulation in a synchronized way. This protocol begins with the insertion of a device impregnated with progesterone that must remain in the vaginal fundus of the cow for a specific period of time. This report aims at describing the removal of a progesterone impregnated implant that was found in the abdominal cavity of a heifer. During a routine reproductive service in a rural property, a recently acquired Jersey heifer, with history of having been submitted to FTAI, was evaluated for pregnancy diagnosis. At the exam, it was observed that the heifer was not pregnant and presented an ovarian follicular cyst. The heifer was then submitted to FTAI again, where an implant was inserted and removed. This made the cyst to recede, and upon subsequent insemination, the applicator was not able to pass the cervix. Twenty days after insemination, the heifer was in estrus and when it was evaluated for insemination, a loose implant was identified in the abdominal cavity of the heifer. A laparotomy was performed through the left flank and the implant was removed. After recovery from the surgery, the heifer was inseminated again and became pregnant. The FTAI protocols should only be carried out by trained professionals to avoid problems such as the one presented herein, where an implant was found in the abdominal cavity of a heifer.(AU)

La inseminación artificial a tiempo fijo (FTAI) es una biotécnica con numerosas ventajas para la producción de ganado. Para realizar FTAI, las hembras bovinas deben recibir un protocolo farmacológico que induzca la ovulación de forma sincronizada. Este protocolo comienza con la inserción de un dispositivo impregnado con progesterona que debe permanecer en el fondo vaginal de la hembra durante un tiempo específico. Este informe tiene como objetivo describir la extracción de un implante impregnado de progesterona que se encontró en la cavidad abdominal de una novilla. Durante la atención reproductiva de rutina en una propiedad rural, se evaluó el diagnóstico de preñez de una novilla de Jersey recién adquirida con antecedentes de someterse a FTAI. En el examen, la novilla no estaba embarazada y tenía un quiste folicular ovárico. La novilla fue nuevamente sometida a FTAI, en la cual se insertó y removió un implante, con esto el quiste retrocedió y al inseminar a la novilla no fue posible pasar el cérvix con el aplicador. Veinte días después de la inseminación, la vaquilla manifestó estro y cuando se evaluó para una nueva inseminación, se identificó un implante suelto en la cavidad abdominal de la vaquilla. Se realizó una laparotomía a través del flanco izquierdo y se extrajo el implante. Después de recuperarse de la cirugía, la novilla fue inseminada y nuevamente quedó embarazada. Los protocolos FTAI deben ser realizados por profesionales capacitados para evitar problemas como la situación actual donde se encontró un implante en la cavidad abdominal de una novilla.(AU)

Testicular shape, scrotal skin thickness and testicular artery blood flow changes in bulls of different ages.

The aim of this study was to compare the biometric testicular characteristics, skin thickness and haemodynamics of the testicular artery of 12- and 24-month-old bulls using Doppler ultrasonography, the study was conducted using 48 indicus-taurus animals. The scrotal circumference (SC) and biometry characteristics of the bulls were measured to calculate the testicular volume. Doppler ultrasonography was used to obtain the haemodynamic values of the testicular artery. The skin thickness and volume were lower (p<.01) in the younger bulls (12 months:4.68 ± 0.68 mm; 168.76 ± 47.96 cm3 ) versus 24 months (5.05 ± 0.89; 499.73 ± 129.24 cm3 ) animals (p<.01). During diastole, mean velocity was lower in the 12 months (7.98 ± 3.83) than in the 24 months (11.37 ± 4.15) animals (p <.05). The 12-month-old animals had higher pulsatility and resistivity indices (0.49 ± 0.02; 0.51 ± 0.20) compared to the 24-month-old animals (0.32 ± 0.16; 0.40 ± 0.15) (p < .05). The final testicular end velocity was lower in animals with long/moderate-shaped (L/M) (7.31 ± 2.91) than in those moderate/oval-shaped (M/O) (11.48 ± 3.88) testicles (p < .05). Animals with L/M testes presented higher pulsatility values and resistivity indices (0.51 ± 0.05; 0.55 ± 0.04) compared to animals with M/O shape (0.29 ± 0.20; 0.36 ± 0.15). We showed that the blood flow of the supra testicular artery between the two evaluated ages differed, and that 24-month-old bulls presented better thermoregulation capacity. Animals with a long/moderate testicular format presented a greater vascular resistance, which was imposed on the blood flow due to the anatomical differences in the testicular artery, resulting in lower velocity, and indicating better heat dissipation in this format.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Synchronization of stage of follicle development before OPU improves embryo production in cows with large antral follicle counts.

In the present study, there was an evaluation of in vitro embryo production (IVEP) in Bos indicus donor cows with small or large antral follicle counts (AFCs) when there was synchronization of follicular dynamics among cows before ovum pick-up (OPU). Donor cows classified as having small or large AFC were submitted to OPU/IVEP program (Experiment-I) or had follicular-stage synchronization imposed before OPU/IVEP (Experiment-II). In Experiment-I, the cows with a large AFC had a greater (P < 0.01) mean of embryos developing to the blastocyst stage compared to those with a small AFC. In Experiment-II, percentage of viable oocytes/OPU were not affected (P = 0.33) by synchronization of follicular dynamics, but the AFC had an effect (P < 0.0001). There was an interaction (P = 0.01) indicating the larger AFC, with or without imposing of a synchronization treatment regimen, resulted in the most desirable outcome. The number of embryos was affected (P < 0.001) by follicular-stage synchronization and AFC, with there being an interaction (P = 0.002) with the most desirable results for the large AFC-synchronized group. Number of pregnancies was greater (P ≤ 0.02) for recipient females with embryos from synchronized donors and with a large AFC. There was an interaction (P = 0.03) with there being a greater pregnancy percentage for cows with synchronized follicular stages and the large AFC. Bos indicus donor with a large AFC when associated with the synchronization of stage of follicular dynamics pre-OPU results in improvement of the efficacy of IVEP.

Follicular wave synchronization prior to ovum pick-up.

Among the reproductive biotechnologies, in vitro embryo production (IVEP) is an important tool for multiplying genetic material of superior merit. Recently, the number of embryos produced and transferred in vitro became significantly higher than that produced in vivo worldwide. In this context, the enhancement was attributable to ovum pick-up (OPU). With the advent of genomic technology, shortened breeding intervals, and increased selection accuracy, IVEP has attracted increasing attention for commercial use. The IVEP technique is well-established, but the embryo production rate has reached a plateau at 30-40%. Despite constant advances, the OPU/IVEP programs face some challenges that hinder the efficient application of the technique. Previous studies have shown that the quantity and quality of aspirated oocytes are essential factors for successful IVEP. This paper presents a brief overview of alternatives that can be employed to improve the process-seeking methods that assist in the recovery of better-quality oocytes and higher competence in OPU to improve embryo production. These strategies include using follicular wave synchronization prior to OPU, employing the influence of antral follicle populations, using the pre-OPU gonadotrophic stimulus and applying non-hormonal methods for selecting female donors.

Influence of age and ovarian antral follicle count on the reproductive characteristics of embryo donor mares.

BACKGROUND: Age and antral follicle count (AFC) are related to fertility in cattle, but this information remains limited in mares. METHODS: To verify the influence of age and AFC on the reproductive characteristics of mares, 15 Quarter Horse donors, with 5-15 antral follicles, ranging from three to 17 years old, healthy and in good nutritional status, were divided into groups with low AFC (≤9 follicles) or high AFC (≥10 follicles) and mares considered young (≤9 years) or old (≥10 years). Mares were submitted to ultrasonography to determine the dominant follicle diameter, follicular growth rate, degree of uterine oedema and embryonic recovery for a minimum of three oestrous cycles. RESULTS: AFC was higher (P=0.001) in young mares compared with old mares. An interaction (P=0.001) between AFC and age was observed with regard to follicular growth rate, being that mares with low AFC and are old showed the lowest follicular growth compared with those of low AFC and young, high AFC and old, and high AFC and young. Younger mares and those with high AFC exhibited higher degree of uterine oedema (P<0.05) on the third day of oestrus compared with older mares and with low AFC (3 and 2, respectively). However, in both groups, the degree of oedema reduced by the time of ovulation. CONCLUSION: Follicular growth rate, degree of uterine oedema and the number of antral follicles are higher in young mares with high AFC. However, the rate of embryonic recovery is not influenced by donors' age or AFC.