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Contents Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized ( IVF) and parthenogenetic ( PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation ( IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock. [ABSTRACT FROM AUTHOR]

Global warming poses significant challenges to the fertility of tropical dairy cattle. One promising approach to mitigate heat stress effects on reproductive function and reduce the carbon footprint is the use of integrated livestock-forest (ILF) systems. The aim of this study was to investigate the effects of two different systems, namely Full Sun (FS) and ILF, on maternal hyperthermia and oocyte quality of Holstein and Girolando heifers during the tropical summer season. The temperature-humidity index (THI) data revealed intense heat stress during the experiment. Both the system (P<0.01) and the breed (P<0.01) factors had a significant impact on vaginal temperature, being hyperthermia more pronounced in the FS system and in the Holstein breed. Over the five time points collected at a 33-day interval, we observed distinct patterns for ILF (P=0.65) and FS (P<0.001) systems, suggesting an adaptive response in animals kept in FS systems. Furthermore, oocyte quality assessment revealed an effect of the system for oocyte diameter (P<0.001) and levels of IGFBP2 (P<0.001), and caspase 3 levels showed a decrease in ILF compared to FS for both Holstein (P<0.001) and Girolando (P<0.001) breeds. Collectively, these parameters indicate that oocyte quality during the summer months was superior in animals maintained in the ILF system. In conclusion, the ILF system demonstrated promising results in attenuating maternal hyperthermia and mitigating its effects on oocyte quality. Additionally, our observations suggest that animals in the FS system may exhibit an adaptive response to heat stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Zinc oxide nanoparticles (ZnO-NPs) have garnered significant attention in biological applications due to their known antioxidant properties. However, their potential impact on assisted reproduction techniques remains largely unexplored, particularly in the context of oocyte quality maintenance within in vitro culture systems, where free radicals can exert detrimental effects. This study investigated the effects of incorporating ZnO-NPs to in vitro maturation (IVM) media on the developmental, cryosurvival, and metabolic profiles of bovine embryos. Three concentrations of ZnO-NPs (0, 1.0, and 1.5 μg/mL) were evaluated. We observed, for the first time, that the inclusion of ZnO-NPs at a concentration of 1.0 μg/mL led to a significant increase in the number of embryonic cells (p < 0.05) accompanied by a reduction in reactive oxygen species production (p < 0.05). Notably, ZnO-NPs did not alter embryonic development, cryosurvival rates, or mitochondrial viability. These findings suggested that ZnO-NPs has antioxidant properties and are compatible with bovine oocytes. Consequently, they may serve as promising supplements to the IVM media, potentially enhancing the efficiency of assisted reproduction techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study aimed to assess the cortisol, body and reproductive development of prepubertal Holstein and Holstein-Gir ¾ heifers at 27 months of age maintained in an integrated livestock-forest (ILF) system for 60 summer days compared to the monoculture system in full sun (FS). The ILF system promoted changes ( P =0.02) in the cortisol levels of Holstein-Gir ¾ heifers and did not affect weight gain in any of the breed groups studied. Animals in ILF system presented a lower ( P =0.006) vulvar development for the rima height parameter and similar for the vulva width parameter. The ovarian follicular population of Holstein-Gir ¾ heifers in the ILF system was lower ( P =0.004); however, for the Holstein heifers, no statistical difference was found, and numbers were higher ( P =0.08) in the ILF system. None of the other ovarian parameters studied had any changes, and we also found important racial differences. Weight gain ( P =0.003), vulvar development ( P <0.001), and mean follicular size ( P =0.008) were higher in the Holstein-Gir ¾ animals. Based on such results, the effect of the ILF system at 27 months of age on stress and reproductive parameters in the Holstein breed is considered positive, although negative effects have been detected on reproductive parameters in the Holstein-Gir ¾ breed., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

Genomic selection has transformed the livestock industry, enabling early-life selection of animals. Biopsy sampling of pre-implantation embryos has been described since 1968. However, it was only after 2010, with the advancement of molecular biology techniques such as whole genomic amplification and SNP Chips, that next-generation sequencing became commercially available for bovine embryos. It is now possible to make decisions about which embryos to transfer not only based on recipients' availability or embryo morphology but also on genomic estimates. This technology can be implemented for a wide spectrum of applications in livestock. In this review, we discuss the use of embryo biopsy for genomic selection and share our experience with Gir and Girolando Brazilian breeding programs, as well as future goals for implementing it in Brazilian bovine in vitro embryo production practices., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

The aim of this study was to establish a platform for genomic selection of in vitro-fertilized (IVF) Gir embryos. Multiple displacement amplification (MDA)-based embryo biopsy samples were genotyped, and genomic estimated breeding values (GEBV) for milk yield (305MY) were calculated. The concordance of GEBV and accuracy between embryo biopsies and the respective liveborn were assessed. Imputation was performed using two panels (Z-Chip and Bovine HD, Illumina) based on a database of 73,110 lactating cow's database and pedigree files from 147,131 animals. Biopsied embryos had similar pregnancy rates (39% vs 40%), pregnancy loss rates (18% vs 20%), and pregnancy length compared to Control embryos. After genotyping, low call rate means were detected for biopsy samples compared to the respective calf samples (0.80 vs 0.98). Imputation presented 0.83 (Z-Chip) and 0.96 (HD) accuracy (CORRanim). Embryo GEBV accuracy levels were higher in BovineHD imputation (0.82) than Z-Chip imputation (0.55) or no imputation (0.62), and the correlation between embryo/calf pairs' accuracy was 0.85 for BovineHD imputation, 0.11 for Z-Chip imputation, and 0.02 for no imputation. GEVB estimates correlation between embryo/calf pairs was 0.87 for BovineHD imputation, 0.80 for Z-Chip imputation, and 0.41 before imputation. The call rate of embryo samples did not affect the correlation between embryo/calf pairs for accuracy and GEBV before and after BovineHD imputation. Embryos obtained on the same farm presented GEBV 305MY differences of up to 800 kg, emphasizing the expected impact of embryo genomic selection for the Gir breed., Competing Interests: Conflict of interest The authors declare that this scientific work was conducted following the ethical guidelines of research and there is no conflict of interest., (Copyright © 2023 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Cattle productivity in tropical and subtropical regions can be severely affected by the environment. Reproductive performance, milk and meat production are compromised by the heat stress imposed by the elevated temperature and humidity. The resulting low productivity contributes to reduce the farmer's income and to increase the methane emissions per unit of animal protein produced and the pressure on land usage. The introduction of highly productive European cattle breeds as well as crossbreeding with local breeds have been adopted as strategies to increase productivity but the positive effects have been limited by the low adaptation of European animals to hot climates and by the reduction of the heterosis effect in the following generations. Gene editing tools allow precise modifications in the animal genome and can be an ally to the cattle industry in tropical and subtropical regions. Alleles associated with production or heat tolerance can be shifted between breeds without the need of crossbreeding. Alongside assisted reproductive biotechnologies and genome selection, gene editing can accelerate the genetic gain of indigenous breeds such as zebu cattle. This review focuses on some of the potential applications of gene editing for cattle farming in tropical and subtropical regions, bringing aspects related to heat stress, milk yield, bull reproduction and methane emissions., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n = 40), average (0.17-0.48, n = 168), and low (<0.17, n = 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p < 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p < 0.05) in high efficiency donors and a decrease (p < 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p = 0.017) by donor's efficiency (low: 0.60 ± 0.09 $$ 0.60\pm 0.09 $$ , average: 1.17 ± 0.07 $$ 1.17\pm 0.07 $$ , high: 2.57 ± 0.26 $$ 2.57\pm 0.26 $$ ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs., (© 2023 Japanese Society of Animal Science.)

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower ( P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower ( P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased ( P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower ( P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro -fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher ( P < 0.05) with 2.5 μM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 μM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 μM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher ( P < 0.05) than for 0 μM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 μM RA33 was compared with 0.5 μM resveratrol, similar ( P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher ( P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower ( P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro -fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

Traditional breeding techniques, applied incrementally over thousands of years, have yielded huge benefits in the characteristics of agricultural animals. This is a result of significant, measurable changes to the genomes of those animal species and breeds. Genome editing techniques may now be applied to achieve targeted DNA sequence alterations, with the potential to affect traits of interest to production of agricultural animals in just one generation. New opportunities arise to improve characteristics difficult to achieve or not amenable to traditional breeding, including disease resistance, and traits that can improve animal welfare, reduce environmental impact, or mitigate impacts of climate change. Countries and supranational institutions are in the process of defining regulatory approaches for genome edited animals and can benefit from sharing approaches and experiences to institute progressive policies in which regulatory oversight is scaled to the particular level of risk involved. To facilitate information sharing and discussion on animal biotechnology, an international community of researchers, developers, breeders, regulators, and communicators recently held a series of seven virtual workshop sessions on applications of biotechnology for animal agriculture, food and environmental safety assessment, regulatory approaches, and market and consumer acceptance. In this report, we summarize the topics presented in the workshop sessions, as well as discussions coming out of the breakout sessions. This is framed within the context of past and recent scientific and regulatory developments. This is a pivotal moment for determination of regulatory approaches and establishment of trust across the innovation through-chain, from researchers, developers, regulators, breeders, farmers through to consumers., (© 2021. The Author(s).)

The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

The present trial evaluated the effect of crossbred composition and Temperature and Humidity Index (THI) on vaginal temperature (VT) of Girolando dairy cows maintained under tropical pasture during warm seasons. The VT was monitored along 41 to 96 h in 615 Girolando cows with different Holstein (H) × Gir genetic composition (1/2 H = 284, 3/4 H = 248, and 7/8 H = 83) from six Brazilian farms in the summer of 2016 and 2017. VT of each cow at each hour of the day and the respective THI were averaged per hour across all monitoring days to generate an averaged value for VT and THI during 24 h. A linear mixed model with repeated measures using restricted maximum likelihood (REML) method for (co)variance components estimation procedure was employed. The final model adjusted the VT for the effects of cow, time, THI, farm, year, pregnancy status, body condition score (BCS), milk yield, genetic composition, and genetic composition*time interaction. Fixed effects were evaluated by ANOVA and tested with Tukey test in R software version 3.6.1 (R Core Team, 2019). Overall mean of VT, air temperature (AT), and THI were 39.06 ± 0.52 °C, 25.63 ± 0.40 °C, and 75.06 ± 3.96, respectively. VT had moderate positive correlation with THI ( r ² = 0.45, P < 0.001) and AT ( r ² = 0.46, P < 0.001). The VT had estimated linear increase of 0.05 °C for each THI unit increase ( P < 0.001). Least square mean of VT varied among the farms ( P < 0.001), pregnancy status ( P < 0.001), and BCS ( P < 0.05) but not for Milk yield ( P > 0.05). The daily average VT was affected by genetic composition ( P < 0.001) with highest temperature for 3/4 H (39.08 ± 0.06 °C a) and 7/8 H (39.09 ± 0.06 °C a) and lowest temperature for 1/2 H (38.95 ± 0.06 °C b). The difference of VT among the three crossbred groups varied in function of the time of the day, from 12:00 to 20:00 h ( P < 0.001), with 3/4 Holstein and 7/8 Holstein cows reaching similar VT, above to the upper limit 39.1 °C and higher than 1/2 Holstein cows during all this period. In conclusion, Girolando cows are sensitive to heat stress in tropical condition during warm seasons. Moreover, Girolando cows with genetic composition higher than 3/4 Holstein display reduced thermoregulatory efficiency. Therefore, Girolando cows in tropical dairy farms require strategies to mitigate heat stress according to their genetic composition., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

While microRNAs (miRNAs) are a class of non-coding RNAs important for embryo development, the relationship between them and heat stress during oocyte maturation has not yet been established. This study investigated the effect of heat shock during in vitro maturation (IVM) on the abundance of bta-miR-20a, -27b, -103, -21-5p, -19b, -1246 miRNAs and DROSHA and DICER1 mRNAs, previously reported for being involved in oocyte maturation, response to heat stress and miRNA biogenesis. Oocytes were exposed for 12h to heat shock during IVM, fertilized in vitro and the presumptive zygotes cultured for eight days. The relative quantification of miRNAs and mRNAs was performed by real-time PCR in vitro-matured oocytes and 8-cell stage embryos. Progression of meiosis, embryonic development and apoptotic indices was also evaluated. Heat shock compromised (p < .05) oocyte nuclear maturation, cleavage and embryo development, with a higher (p < .05) embryonic apoptotic index than the control group. The abundance of bta-miR-19b increased (p < .05) whereas the abundance of DROSHA transcripts decreased (p < .05) in embryos derived from heat-shocked oocytes. In conclusion, heat shock during IVM influences the abundance of bta-miR-19b and DROSHA in pre-implantation embryos, indicating a persistent effect of heat shock that can be associated with impaired embryo development., (© 2021 Wiley-VCH GmbH.)

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Artificial reproduction in dairy cattle is challenged by summer temperatures in tropical environments. We describe a treatment based on mild temperature increases to induce thermotolerance and improve the embryo's performance under heat stress conditions. A protocol was established to induce upregulation of heat shock protein A (HSPA, formerly known as HSP70) but not impair embryonic development. Thermal treatment (TT) had no effect on morula/blastocyst rate or blastocyst quality (cell number and apoptosis). Heat shock given one day after TT revealed higher (P=0.00) survival rates in TT blastocysts compared with Control. Treated embryos were transferred to recipients and no detrimental effects were observed regarding pregnancy rates, length, fetal growth or calf weight. Our results demonstrated that the established TT protocol could induce a thermal response by the embryo and is safe for further development.

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study evaluated the role of progesterone (P 4 ) and medroxyprogesterone acetate (MAP) on the molecular status of immature cumulus-oocyte complexes (COCs) and the implications for oocyte quality in sheep. The number of viable COCs per ewe and the rate of COCs screened for developmental competence by brilliant cresyl blue positive (BCB + ) were similar (P > 0.05), respectively, across treatments (P 4 : 7.7 ± 0.7 and 4.7 ± 1.2; MAP: 5.7 ± 1.0 and 3.5 ± 2.3; and control: 5.7 ± 1.1 and 3.6 ± 2.4). The COCs' gene expression was altered by exogenous progestogens compared with the control group: markers of steroidogenic pathway (FSH receptor [FSHr], LH receptor [LHr], and estradiol receptor α) and of quality (zygote arrest 1, growth differentiation factor 9, and B-cell lymphoma 2) were in abundance in P 4 (P < 0.05). In addition, reelin protein (RELN) was downregulated, and Bcl-2 was upregulated in MAP (P < 0.05). In the P 4 vs MAP comparison, FSHr, LHr, and RELN genes were upregulated (P < 0.05) in the P 4 group. In conclusion, P 4 and MAP promoted dissimilar effects on transcriptome profiling of immature BCB-selected COCs, possibly due to the differences in the chemical structure of progestogens and concentrations of serum P 4 . Exogenous P 4 impacted positively on the profile of genes related to oocyte quality., (Copyright © 2020. Published by Elsevier Inc.)

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts., (Copyright © 2020 Camargo, Owen, Van Eenennaam and Ross.)

This study aimed to evaluate chromium supplementation on productive, reproductive, and metabolic parameters at lactating Girolando cows subjected to heat stress conditions in a climatic chamber. Thirty-six lactating Girolando cows were subjected to two sequential trials. In trial 1 (thermoneutral environment), the effect of chromium supplementation was evaluated (0 vs. 0.50 mg/kg of dry matter). In trial 2, the cows were fed the same diets, but they were divided into three environmental conditions: heat stress conditions in climatic chamber, fed ad libitum (HS); thermoneutral environment, fed ad libitum (TN); and thermoneutral environment, pair-fed (PF). In thermoneutral conditions, chromium supplementation did not affect productive or metabolic parameters, although supplemented cows had lower viability of oocytes (65.11 ± 0.08% vs. 76.86 ± 0.08%). During heat stress, chromium supplementation lowered plasma glucose levels (61.17 ± 1.90 vs. 67.11 ± 1.90 mg/dL), and increased the insulin:glucose ratio (0.39 ± 0.04 vs. 0.27 ± 0.04). Cows fed the control diet in the HS group had higher vaginal temperature values (39.40 ± 0.10 °C) than the cows in the TN group and PF group (38.89 ± 0.10 °C and 38.85 ± 0.11 °C, respectively). However, supplemented cows heat-stressed maintained the same vaginal temperature as cows in thermoneutral conditions. In conclusion, chromium supplementation improved glucose metabolism and prevented body temperature increases under heat stress conditions.

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA-dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA-dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA-dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA-dLCr 1:25 and the controls. The DNA-dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA-dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA-dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

This study aimed to evaluate the effect of two Embryo Manipulation Solutions (EMS and EMS supplemented) in maintenance of the viability of embryos, initially using structures derived from mice (first phase). Next, the efficiency of these solutions in routines of bovine embryo transfer was evaluated (second stage). Mice embryos were used in the stages of early blastocyst, and compact morula grades I and II. These embryos were initially randomly distributed and maintained for four hours in three solutions: Modified phosphate buffered saline (PBS; Control); EMS (treatment 1), and EMS supplemented (treatment 2). Subsequently, they were cultured in TCM 199 medium and evaluated in terms of total number of cells, morphometric characteristics, ultra structural aspects, detection of cell apoptosis, and quantification of Hsp 70.3 gene expression. In the second phase, these same solutions were tested in the transfer of quality I and II bovine embryos (excellent and good). These embryos were transferred fresh to 58 recipients. The results showed that the total number of cells in embryos expanded blastocyst (ExB), the number of apoptotic cells, the cell, nuclear, nucleolar diameter and the nucleus/nucleolus ratio was similar among the treatments. The pregnancy rate shown on second phase was also similar. However, the EMS supplemented expressed more Hsp70.3 than EMS. The expression of Hsp70.3 was also greater for embryos in EMS than that of EMS supplemented. The McII embryos, EMS and EMS supplemented samples also expressed more Hsp70.3 compared to control embryos. In conclusion, the tested solutions can be used in routine embryo transfer techniques, replacing modified PBS solution as an effective media in maintaining embryo viability., (Copyright © The Author(s). Published by CBRA.)

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192 hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p < .01) and up-regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes., (© 2019 Blackwell Verlag GmbH.)

Heat stress compromises bovine oocyte developmental competence, but the effects of high temperature during oocyte maturation on embryo chromatin organization is unknown. In this study bovine oocytes were exposed to heat shock (41°C) for 12 h during in vitro maturation and then submitted to in vitro fertilization. The heat shock did not affect (P > 0.05) the cleavage but reduced (P < 0.01) the blastocyst rate on Day 7 and Day 8. No effect (P > 0.05) on total cell number was found, but the heat shock increased (P < 0.05) the proportion of apoptotic cells in blastocysts at Day 8. Immunofluorescence analysis of H3K9me3 and HP1 was performed in embryos at 52 h post in vitro fertilization. An accumulation of H3K9me3 in the nuclei of embryos derived from heat-shocked oocytes at four-cell and eight-cell stages was found. Also, a non-expected higher proportion (P < 0.05) of four-cell stage embryos displaying nuclei with increased HP1 fluorescence was observed, suggesting an abnormal chromatin compaction in embryos from heat-shocked oocytes. Embryos at eight-cell stage derived from heat-shocked oocytes displayed lower (P < 0.05) relative amount of HSP40 transcripts than control ones. In conclusion, heat shock before fertilization has an effect on embryo chromatin, influencing the accumulation of H3K9me3 and HP1 in early embryos as well as further development.

Most early developmental data are lost in bovine embryo culture systems. We developed and validated a method for culture of bovine embryos in groups that allow individual assessment. An autoclavable low-cost multiembryo chamber (MEC) was prepared using a polyester mesh fixed to a glass coverslip. Embryonic development was not affected by MEC. Compared to conventional bovine culture system (oil-covered drops, control), cleavage (C, 71.2 ± 7.8%; MEC, 74.3 ± 6.0%), blastocyst rate (C, 29.9 ± 4.4%; MEC, 28.3 ± 5.0%) and blastocyst cell number (C, 94.1 ± 9.7; MEC, 92.9 ± 5.3) were similar. Caspase 3 positive cell index in blastocysts was increased in MEC group, but apoptosis rate was below 5% (C, 2.9 ± 0.5; MEC, 4.6 ± 0.6). Using MEC, we performed a retrospective analysis for 'failure' and 'success' embryos, based on their ability to reach the blastocyst stage. We detected the majority of 'success' embryos displayed 8 cells at 48 h post-insemination (hpi) (48.7%), but blastocysts derived from this pattern presented lower cell numbers (91.3 ± 4.2 vs. 107.9 ± 4.9) and higher apoptosis index (6.2 ± 0.6 vs. 4.4 ± 0.5) than blastocysts from 4-cell embryos at 48 hpi. Most (72.0%) embryos that were at morula stage 120 hpi reached blastocyst stage at 168 hpi. Those blastocysts presented more number of cells than blastocysts derived from embryos exhibiting 16 cells at 120 hpi (108.6 ± 4.1 vs. 83.9 ± 4.8). Combination of embryo kinetics data at 48 and 120 hpi revealed high chances of blastocyst formation for patterns: 8 cells/morula, 4 cells/morula, 8 cells/16 cells and 4 cells/16 cells. Blastocysts formed from 4-cell/morula and 8-cell/morula patterns represented 69% of all 168 hpi blastocysts. Blastocysts derived from 4 cells/16 cells displayed decreased apoptosis (3.1 ± 0.6). Our results suggest that MEC can be used for bovine embryo culture without detrimental effects on development and can help to predict blastocyst formation and quality of in vitro fertilization (IVF) embryos. Abbreviations: BSA: bovine serum albumine; COC: cumulus-oocyte complex; FERT-TALP: Tyrode's albumin lactate pyruvate fertilization; FBS: fetal bovine serum; IVF: in vitro fertilization; MEC: multiembryo chamber; PBS: phosphate buffered saline; SOF-AA: synthetic oviductal fluid with amino acids medium; TCM: Tissue Culture Medium.

Objective: In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors., Methods: A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo -matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR., Results: Oocytes in the IN VITRO-OPU group had lower ( p <0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups ( p <0.05)., Conclusion: In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.

Despite the progress on development of new culture media, in vitro-produced embryos still display lower quality when compared to the in vivo-produced counterparts. Coculture has been reconsidered as an alternative to improve embryo quality. Mesenchymal stem cells (MSC) and murine embryonic fibroblasts (MEF) have been extensively used as feeder layers due to their capacity to release growth factors. In the present study we investigated the effect of these feeder layers in oocyte maturation and/or embryo development under in vitro conditions. Oocytes were matured in control (CTRL) conditions or in coculture with MSC or MEF. In vitro fertilization and embryo culture until fourth day were performed in CTRL condition for all groups. Embryos from fourth day on were then cultured until the eighth day in CTRL or in coculture system. No significant differences for metaphase II stage and apoptosis in oocytes were found among the groups. There was also no difference among the groups when we evaluated blastocyst formation on the seventh and eighth day, with exception of a higher hatched blastocyst rate in the group maturated and cultivated in CTRL condition when compared to the group matured and cocultured with MSC. Also no difference was observed in the number of cells in the whole embryos, in the inner cell mass, in the trophoblast and at apoptotic stage on the eighth day. We conclude that coculture with MSC or MEF during maturation and/or embryo development do not enhance the in vitro production of bovine embryos.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

l-carnitine is an antioxidant and β-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of the present study was to characterise the roles of intrafollicular oestradiol production and granulosa cell (GC) expression of the LH receptor (LHR) gene and its isoforms during follicular deviation in Bos indicus. Follicular wave emergence was synchronised in heifers from a Bos taurus dairy (Holstein; n=10) and a B. indicus dairy breed (Gir; n=10). Follicles were aspirated individually at sizes corresponding to the periods of predeviation, deviation and postdeviation. Intrafollicular oestradiol (IF-E2) and progesterone (IF-P4) concentrations were determined in the follicular fluid (FF) by radioimmunoassay, and relative expression of P450 aromatase (CYP19A1) and LHR forms was evaluated in GC using real-time quantitative-polymerase chain reaction. Despite differences in the size of the dominant follicle at deviation, changes in CYP19A1 expression and IF-E2 concentrations were similar in follicles of the same diameter in both breeds. A peak in total LHR expression occurred after follicular deviation in association with low expression of LHR isoforms. The results suggest that regulation of LHR function by sequential changes in the expression pattern of LHR isoforms may play a role in the early deviation of the dominant follicle, as observed in B. indicus breeds.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF-induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC., (Copyright © 2016 Elsevier Inc. All rights reserved.)