Introduction. Vulvovaginal candidiasis (VVC) is a common fungal infection that has a significant impact on global public health. Although studies associate VVC with male infertility, its influence on the female reproductive system, particularly uterine involvement, remains unknown.Gap statement. While recent animal studies propose that Candida albicans migration from the vaginal tract to the uterus in VVC could lead to infertility, the underlying histopathological alterations that support this connection are not well understood.Aim. To investigate possible changes in the uterine tissue of BALB/c mice infected experimentally with C. albicans, by analysing the progression and effects of infection on the uterus.Methodology. Female BALB/c mice were divided into two groups (infected and control). Vaginal infection was induced by C. albicans, and vaginal and uterine tissues were collected at different intervals (1, 3, 5, 7 and 10 days). Analyses included fungal burden (c.f.u. g-1), histopathology stained with Grocott-Gomori and macroscopic and microscopic evaluation (haematoxylin-eosin staining) of uterine tissue.Results. Vaginal infection was confirmed by a consistent presence of yeast in vaginal tissue. C. albicans migration was observed in the uterus, with a significant increase in fungal burden on day 3, followed by macroscopic alterations such as oedema and hyperaemia. Histologically, inflammatory infiltrates, epithelial necrosis and progressive degeneration were identified until day 7, with signs of resolution by day 10.Conclusion. The results demonstrate that vaginal infection by C. albicans was able to cause significant uterine alterations with self-limiting progression. These findings suggest that VVC may have direct implications for female fertility, warranting future investigations into its influence on infertility cases.

We evaluated the efficiency of two-dimensional (2D) and three-dimensional (3D) systems for in vitro culture of preantral follicles from the bovine species. Ovarian cortex fragments were obtained from a local slaughterhouse and randomly distributed into four groups: i) standard culture, directly on a culture plate (2D), ii) supported culture, on a gel monolayer (2D), iii) immersed culture on agarose gel (plating coated) covered in a medium (3D) and iv) Millicell-Biopore culture (3D), tested at 6 or 14 days. The following parameters were evaluated: follicular morphology and morphometry, reactive oxygen species production and granulosa cell proliferation. After 6 days of culture, follicles cultured on a gel monolayer (2D) showed a higher proportion of intact follicles than those cultured using other methods (P < 0.05). After 14 days of growth, standard culture on a culture plate (2D) and supported culture on a gel monolayer (2D) had a higher percentage of morphologically intact follicles (P < 0.05) than the 3D culture. An assessment of the redox status revealed that superoxide anion levels and antioxidant capacity of the samples cultured using the four methods were lower than those of the control group (non-cultured; P < 0.05). Taken together, these findings suggest that the agarose gel support system offers distinct advantages for bovine preantral follicle culture by maintaining morphological integrity and stabilising the redox environment, although further studies incorporating functional endpoints are warranted to fully confirm these observations.

Context Adequate progesterone levels are crucial for embryo development and maintenance of pregnancy. Aims This study aimed to evaluate the effects of injectable progesterone (iP4) administered 14 days after timed artificial insemination (TAI) on conception at Day 30, maintained pregnancy at Day 60 and pregnancy loss in high-yielding dairy cows. We hypothesized that exogenous iP4 supplementation during the critical period of maternal recognition of pregnancy would enhance conception rates. Methods A total of 382 lactating Holstein cows were subjected to TAI. On Day -11, they received an intravaginal device containing 1.9 g of P4, associated with 10.5 mu g of buserelin acetate (gonadotropin-releasing hormone) and 2 mg of estradiol benzoate. On Days -4 and -2, the animals received 25 mg of dinoprost tromethamine, and on Day -2, the device was removed and 1 mg of estradiol cypionate was administered. On Day 0, TAI was performed. Fourteen days after insemination (Day 14), the cows were divided into two groups: control (n = 186) and iP4 (n = 196, supplemented with 350 mg iP4). Pregnancy diagnosis was performed on Days 30 and 60 post-TAI. The data were analyzed using a mixed-effects model that included fixed effects, random effects and covariates. Key results The conception rates at 30 and 60 days post-insemination were significantly higher (P < 0.05) in the iP4 group (44.7% and 39.2%, respectively) than in the control group (35.1% and 31.7%, respectively). A significant interaction was observed between iP4 supplementation and body condition score, in which cows with body condition scores >2.75 treated with iP4 had a higher conception rate at 30 and 60 days than those in the control group. Pregnancy loss between 30 and 60 days was not affected by treatment. Implications These findings suggest that iP4 supplementation 14 days after artificial insemination may improve fertility in high-producing dairy cows, but they should be interpreted with caution due to the lack of physiological evaluation of the mechanisms underlying the action of exogenous progesterone.

The objective of this study was to evaluate the effects of gonadotropin-releasing hormone (GnRH) administration at the beginning of a fixed-time artificial insemination (FTAI) protocol on ovarian dynamics and haemodynamics, assessed by Doppler ultrasonography, in Holstein cows. Sixteen lactating cows were enrolled and allocated to two experimental groups. After pre-synchronisation, the number of corpora lutea (CL) was quantified, and blood samples were collected for progesterone (P4) measurement at the beginning of the FTAI protocol (day 0; D0). The control group (n = 8) received 5 mL of 0.9% NaCl solution intramuscularly, whereas the GnRH group (n = 8) received 21 & micro;g of buserelin acetate (GnRH analogue). On day 7 of the protocol (D7), ovarian ultrasonographic examination was repeated, and additional blood samples were collected. On day 10 (D10), the diameter and blood perfusion of pre-ovulatory follicles (POFs) were measured. On D0, there was no difference in the number of CLs or P4 levels, whereas on D7, GnRH increased the number of CLs (2.25 +/- 0.17 vs 1.12 +/- 0.17; p = 0.005) and P4 levels (12.22 +/- 0.52 vs 9.38 +/- 0.52 ng/mL; p = 0.001) compared with the control group. On D10, POFs in the GnRH group showed a greater blood perfusion area (16.74 +/- 3.41 vs 7.52 +/- 1.15 mm2; p = 0.031). The use of GnRH on D0 of the FTAI protocol resulted in a significant increase in the number of CLs, P4 levels, and blood supply to pre-ovulatory follicles.

We tested the effects of centrifuging in vitro matured bovine oocytes for varying times on embryo development and cryotolerance. The oocytes were divided into four groups: control (GC) and centrifuged groups [5433 x g: G5, n = 463 (5 min); G10, n = 461 (10 min); and G15, n = 483 (15 min)]. After centrifugation, the oocytes underwent in vitro fertilization for embryo production. Two parameters were evaluated: i) embryonic development (n = 1,878), and ii) cryotolerance evaluation (survival and hatching rates; n = 303). The CG and G10 groups showed blastocyst rates of 42.25% and 45.77%, respectively, higher than those of the other groups (p = 0.02). The hatching rate was equal (p > 0.05) in CG (91.96%), G5: (87.74%), and G10: (95.73%) groups; however, it was lower in G15: 77.06% (p < 0.01). In the CG group, 65.88% of cryopreserved embryos survived, which was different (p < 0.05) from that in G5 (82.02%) and G10 (82.28%) (p > 0.05). Post-freeze hatching percentage was 74.0%, 87.7%, and 47.7%, in G5, G10, and G15, respectively, which was significantly greater than that in CG (p < 0.01; 26.8%). Post-freeze hatching percentage in only G10 matched that of the non-cryopreserved embryos CG (p = 0.06, 92%). We conclude that oocyte centrifugation for 10 minutes was efficient for in vitro embryonic development and cryopreservation of cattle embryos.

This study compared the reproductive performance of three different programs using conventional fixed-time artificial insemination (FTAI), fixed-time embryo transfer (FTET), and their combined use, considering estrus intensity as a criterion for the reproductive program. Brangus multiparous cows (n = 1.100), 40-50 days postpartum, 4-8 years old, and body condition scores 2.50 of 4.50 underwent a protocol for ovulation synchronization based on progesterone (P4) and estradiol on D0. On D10, one of three Programs I-III was implemented: control FTAI (n = 147 cows), control FTET (n = 617) with embryos from in vitro production (IVP) on D17, or FTAI + FTET (n = 336), where cows with low or no estrus expression were inseminated on D10, similar to Program I, while those with high-intensity expression received one embryo from IVP on D17, similar to Program II. Corpus luteum (CL) size and quality were assessed using B-mode and Doppler ultrasound on D17. The service/utilization rate was higher for FTAI and combined FTAI + FTET than for the conventional FTET. The program that used only FTAI resulted in higher pregnancy at 30 and 60 days, as well as lower pregnancy loss, compared to programs that used embryos. Furthermore, the FTAI + FTET program showed reduced pregnancy loss, compared to the FTET program. In the combined program, the CL was greater in those who received FTET alone than in those who received FTAI alone. A higher proportion of recipients with CL with a high luteal blood perfusion score was observed in the FTET group compared to the FTAI group. Satisfactory reproductive rates can be achieved using conventional FTAI or FTET programs. However, the combined program, associated with monitoring the intensity of estrus expression, is a promising strategy for allocating females with absent or low estrus expression to FTAI, and those with high estrus intensity to FTET. Recipients with high-intensity expression had higher CL quality and, when transferred, maintained satisfactory reproductive performance compared with conventional FTET.

Oxidative stress (OS) induced by an imbalance in reactive oxygen species (ROS) levels in vitro impairs embryonic development. Here, we assessed the effects of alpha-lipoic acid (ALA) in in vitro production media on OS reduction, embryonic development, and cryotolerance of bovine embryos. We evaluated the effects of adding different concentrations of ALA (2.5, 5, 10, and 25 μM) to in vitro maturation (IVM) or in vitro culture (IVC) medium on embryonic development. We also determined the effects of adding ALA (25 μM) to the IVM and IVC medium in the same routine on the development and quality of embryos, ROS levels, and cryotolerance. Embryos were produced in vitro using conventional protocols for each treatment. The inclusion of ALA in the IVM and IVC media did not affect the development or quality of embryos; however, it reduced ROS levels in grade II embryos and increased hatching after 12 h on day 7 in grade I embryos and on day 8 in grade II embryos after warming. These findings prompt questions regarding the potential of ALA in improving embryo metabolism, considering the initial embryo recovery in the first few hours of embryo warming.

The postpartum period presents significant reproductive challenges and remains relatively understudied in beef cattle. This study evaluated the effects of prostaglandin F2α (PGF2α) and injectable progesterone (iP4) on uterine health, ovarian dynamics, and pregnancy per insemination in Nelore cows subjected to timed artificial insemination (TAI) during early postpartum. In Study I, cows at 10 days postpartum (dpp) were randomly assigned to one of four groups: Control (0.9 % NaCl, n = 10); PGF2α (0.5 mg cloprostenol, n = 10); iP4 (150 mg, n = 10); or PGF2α+iP4 (0.5 mg cloprostenol and 150 mg iP4, n = 10). Animals were evaluated for uterine inflammation-based on polymorphonuclear cell count (PMN) and uterine body diameter-at 10 and 30 dpp, and underwent an estradiol/progesterone-based TAI protocol to monitor follicular dynamics. In Study II, which was specifically designed to assess pregnancy outcomes, cows at 22 ± 2 dpp were randomized into the same four treatment groups: Control (n = 520), PGF2α (n = 513), iP4 (n = 521), and PGF2α+iP4 (n = 506). At 10 dpp, PMN counts were similar among treatment groups (p = 0.23) but showed a significant reduction by 30 dpp (p < 0.05). No treatment effect was observed at 30 dpp on PMN counts (p = 0.89) or incidence of subclinical endometritis (p = 0.53). Uterine diameter decreased (p < 0.05) in all groups from 10 to 30 dpp, except in the iP4 group. At 30 dpp, the PGF2α and iP4 groups showed a larger dominant follicle diameter compared to controls. PGF2α-treated cows ovulated earlier (p = 0.01), and those receiving PGF2α tended to respond better to the TAI protocol (p = 0.06). In Study II, the PGF2α+iP4 group achieved the highest pregnancy per TAI rate (354/506; 70 %), outperforming the PGF2α (333/513; 64.9 %), iP4 (331/521; 63.5 %), and Control (325/520; 62.5 %) groups (p = 0.05). Treatment with PGF2α+iP4 in early postpartum Nelore cows improved pregnancy rates following TAI.

While antral follicle count (AFC) has been associated with higher pregnancy rates, at present, our understanding of it as a reproductive parameter remains incomplete. This study aimed to characterize gene expression profile of oocytes from crossbred Bos taurus x Bos indicus heifers with high and low AFCs. Crossbred Nelore-Angus heifers (n = 50) with a mean (SD) age of 9.6 +/- 0.55 months, a weight of 295.4 +/- 32.6 kg, and a BCS of 3.44 +/- 0.41 were studied in a feedlot system. The heifers received a hormonal protocol based on injectable progesterone and estradiol cypionate administered 12 days apart, and ovarian ultrasonography (US) was performed 12 days after to assess the AFC. Based on AFC, heifers were divided into low (<= 14 follicles) and high (>= 31 follicles) AFC, groups.Forty-five days after US, 14 heifers were slaughtered, and their ovaries were collected for morphological analysis and follicle aspiration. Cumulus-oocyte complexes (COCs) from the high and low AFC groups were graded according to their quality. Only best-quality COCs were stored for RNA-seq analysis. No differences were found in the presence or diameter of the dominant follicle and corpus luteum in the US, nor in the volume of the dominant follicle postmortem. The quantity of COCs recovered from high-AFC heifers was higher than that from low-AFC heifers (P < 0.05), and a tendency (P = 0.07) toward a higher amount of grade II COCs was observed. Thirty-two genes were differentially expressed between the groups, of which 30 were up-regulated and two down-regulated in the low AFC group. Among these, 22 % (7/32) were associated with fertility (CAB39, SLC2A6, CITED2, FDX1, HSD11B2, CD81, and PLA2G12B). Moreover, 9 and 2 exclusive genes were identified in the high and low AFC groups, respectively. Enrichment analyses showed that genes exclusive to oocytes from low-AFC heifers were associated with fundamental cellular processes, such as biosynthesis/biogenesis of ribosomes, peptides, amides, and nucleotides, and also with autophagy, mitophagy and mTOR signalling pathways.On the other hand, only one pathway was enriched in the high AFC group, but this cannot be related to the events studied No differences were observed in the ovarian structures after pre-synchronization of the estrus cycle of young Crossbred Nelore-Angus heifers. However, a tendency of a higher amount of grade II COCs was observed in heifers with high AFC than in those with low AFC. RNA sequencing results indicated that the main differences between high and low AFC heifers were not reflected in the genes directly related to fertility.

This study aimed to assess differences in the immunostaining intensity of follicle-stimulating hormone receptor (FSHr) and leutenizing hormone receptor (LHr) receptors in the ovarian follicles of Bos indicus cows with high or low antral follicle counts (AFCs). Ovaries from cyclic Nelore cows (N = 20) were obtained from a local slaughterhouse and classified based on AFC (≥ 3 mm) into high- (≥ 30 follicles, N = 10) and low- (≤ 15 follicles, N = 10) AFC groups. Immunohistochemical studies were performed for FSHr and LHr. Immunostaining intensity was measured using ImageJ software with the IHC Profiler plugin, and pixel intensity was measured on a scale of 0 (darkest) to 255 (lightest). An interaction was observed between the AFC group and follicular developmental stage for FSHr immunostaining intensity, with preantral follicles from the low-AFC group showing highest immunostaining intensity (p < 0.0001). The FSHr immunostaining intensity of antral follicles from the low-AFC group was higher than that of the high-AFC group (p = 0.03). LHr immunostaining intensity also was higher in the low-AFC group than in the high-AFC group (p = 0.002). These findings suggest that ovarian follicle characteristics of low-AFC cows have distinct characteristics that could affect their response to reproductive treatments.

Competent oocyte is a critical point for successful fertilization and early embryonic development. This systematic review aimed to synthesize scientific evidence from omics studies on oocyte development in cattle during the estrous cycle. Our systematic search followed the PRISMA guidelines, using three databases: PubMed, Scopus, and Web of Science Core Collection. After a critical appraisal of the literature using the Kmet scoring system, ten peer-reviewed articles were included. These studies involved analyses of follicular fluid, cumulus and granulosa cells, oocytes, and maturation media. Transcriptomics predominated among the studies and identified transcriptional patterns associated with oocyte competence in different cell types and stages of the estrous cycle, particularly around the LH surge. In addition, experiments focusing on the ovulatory window as the main theme were identified in the studies. Metabolomic analyses highlighted the role of amino acid turnover and purine metabolism in follicular fluid and maturation media, providing insights into potential biomarkers in oocyte development. The results emphasized the importance of nucleotide metabolism, extracellular matrix interactions, and hormonal signaling pathways, particularly involving genes such as INHBA, TNFAIP6, and TRIB2, in regulating oocyte maturation. This review underscores the power of integrating omics data to elucidate complex molecular mechanisms relevant to the acquisition of oocyte competence. Furthermore, it explores the identification of potential molecular markers that would aid the development of protocols and culture media, while aiming to improve oocyte quality and advance assisted reproductive technologies.

The objective of this study was to evaluate the effect of adding of C-Type Natriuretic (CNP) to the in vitro culture medium of bovine embryos on cryotolerability through modulation of lipid content and profile, as well as modulation of gene transcripts linked to embryonic metabolism. Initially, a concentration of 400 nM of CNP was used throughout the in vitro culture and blastocysts were collected for lipid content analysis by Sudan Black B. In addition, blastocysts were selected by morphological quality and developmental stage, and the samples collected were analyzed using MRM- profiling. After, blastocysts were vitrified using OPS. Subsequently the warming, hatched blastocysts were collected and evaluated for transcript abundance in a microfluidic platform. Differences of probabilities lower than P < 0.05, and/or fold change ˃1.5 were considered significant. The CNP group presented a reduction in the relative abundance of ions belonging to different lipid subclasses, such as acylcarnitine, sphingomyelin, cholesteryl esters, free fatty acids, and glycerophospholipid. Furthermore, the triacylglycerol lipids TG 52:3 NL 16:1, TG 56:3 NL 18:1, and the glycerophospholipid C22:6, were increased in the CNP group. A modulation of blastocyst transcripts was also observed by increased transcription of ATF4, and a trend statistical significance of BMP15 and GFPT2 transcripts. There was no difference in blastocyst development rates after warming of CNP-treated embryos.

This study aimed to evaluate the effect of prostaglandins on the proportion of inflammatory cells (PMN) and postpartum pregnancy rates in cows subjected to fixed-time artificial insemination (FTAI) programs. In total, 209 postpartum Nelore cows were used in this study. Females were separated into three groups to receive the following treatments: control group- CTL (n = 66), females that were not treated; group 0.5 PG (n = 68), cows that received cloprostenol 0.5mg – a PGF2α analog; and group 1.0 PG (n = 75), cows that received 1mg of cloprostenol. All females were subjected to the FTAI protocol at an average of 38.7 ± 7.6 (Mean ± SD) days postpartum (DPP). Uterine health was assessed using polymorphonuclear cell (PMN) counts. On Day 0 of the FTAI protocol (D0), material was collected using disposable cervical brushes for cytology and analysis of the proportion of inflammatory cells (PMN) recovered from the endometrium in the cervical region. Pregnancy diagnosis was performed by transrectal ultrasonography 30 days after FTAI. Logistic regression was used to analyze the effects of treatment, category, and their interactions on Pregnancy/Artificial Insemination (P/AI). The proportion of PMN cells according to the group was analyzed using ANOVA (PROC GLIMMIX; SAS Inst. Inc., Cary, NC, USA), and the means were compared between groups using Tukey’s test. A level of 5% was considered significant. The proportion of PMN did not differ among the groups. The overall pregnancy rate was 72.2% (151/209). No effects of group (P = 0.51) or category (P = 0.84) were detected on the P/AI among the groups. There was a tendency (P = 0.07) for a group-category interaction for P/AI. In this regard, the P/AI in multiparous cows treated with 1mg of PGF2α tended to be higher (P = 0.08) than that in the control cows.

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.

Pregnancy losses negatively affect the cattle industry, impacting economic indices and consequently the entire production chain. Early embryonic failure has been an important challenge in the embryo industry because proper identification of embryo death at the beginning of gestation is difficult. This review aimed to provide a better understanding on reproductive failure and the relationship between early embryonic loss and different reproductive biotechniques. This review also considers insights and possible strategies for reducing early embryonic loss. The strategies addressed are as follows: i) great impact of rigorous embryo evaluation on reducing embryo losses; ii) selection of recipients at the time of transfer, taking into account health and nutritional status, and classification of the corpus luteum using ultrasound, either in area or vascularization; and iii) paternal effect as one of the factors that contribute to pregnancy losses, with a focus on embryo transfer.

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.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental stage embryos remains to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV), metaphase II (MII), presumptive zygotes (PZ), morula (MO), and blastocyst (BL) stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In the Experiment I, we detected and quantified the NPR2 on the above-mentioned embryo stages. Following, Experiment II, intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e. day 1, or from day 5). In Experiment III, it was used 400 nM of CNP on day 1 (D1) in the IVC, which did no demonstrate to be embryotoxic, and it showed potentially promising results in blastocyst production rate, when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance to competence and quality (apoptosis, oxidative stress, proliferation and differentiation) and lipid metabolism. Differences with probabilities less than P < 0.05, and/or Foldchange (FC) ˃1.5 were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage when there was a significant decrease in membrane receptors. There was no statistical difference in production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (P=0.057), and downregulated transcripts, in BMP15 (P= 0.013), ACAT1 (P= 0.040), CASP3 (P=0.082). In addition, a total of 12 transcriptions in morula presented variation FC ˃1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, HSPA5 transcripts, and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC >1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism and it was not embryotoxic morphologically.

There is a growing regulatory and scientific interest in the studies of environmental substances that are capable of interfering with the reproductive system. Among them, parabens stand out due to their widespread use and frequent detection as contaminants in human tissues and biological fluids. Therefore, we evaluated the toxic effects of butylparaben on the viability and follicular staging of bovine ovarian follicles in vitro. Fragments of ovaries from five cyclic bovine females were cultured for 44 h in a minimal essential medium (MEM; control) or MEM supplemented with 50 µg/mL and 100 µg/mL of butylparaben (BP 50 and BP 100 groups, respectively). The ovarian fragments were subjected to follicular staging, morphological analysis, morphometric analysis, estradiol analysis and oxidative profiling. No significant changes were observed between the experimental groups in follicular staging, estradiol analysis and oxidative profile analysis. However, the BP 50 group showed a significant decrease in the number of intact ovarian follicles. Moreover, a decrease in the follicular and oocyte diameters was observed in the groups that were exposed to butylparaben. In conclusion, butylparaben impairs the integrity and size of ovarian follicles in an in vitro bovine model, but does not affect the oxidative profile and steroidogenesis.

This study evaluated the effect of prostaglandin F2α (PGF2α) associated with gonadotropin-releasing hormone (GnRH) for ovulation induction in precocious indicus heifers submitted to a fixed-time superovulation (SOV) programme. Precocious Nellore heifers (n = 35), aged 13 months, were subjected to the SOV protocol. On day 0 (D0), all animals received intravaginal insertion of a progesterone (P4) device along with intramuscular administration of 2 mg of oestradiol benzoate, plus 200 IU of follicle-stimulating hormone in decreasing doses, with 12-h intervals between D4 and D7, in addition to 150 μg of D-cloprostenol on D6 and device removal on D7. On D8, the donors received 10.5 μg of buserelin acetate and the treatment group received 300 μg of D-cloprostenol/PGF2α. Artificial insemination was performed 12 h and 24 h after GnRH administration using frozen semen. On D15 of the protocol (i.e., D7 after insemination), the embryos were collected and evaluated. All animals passed through the control and treatment groups. Results were evaluated by analysis of variance using an adjusted mixed-effects model (p < 0.05). There was no difference in the total number of embryos between the control and treatment groups (10.40 ± 1.52 vs. 9.60 ± 1.36; p = 0.63) or viable embryos (6.30 ± 1.22 vs. 4.30 ± 0.71). For precocious indicus heifers, treatment with PGF2α in association with GnRH did not affect embryo production in the fixed-time SOV protocol.