Background:Heat shock during sperm capacitation affects the spermatozoa quality, resulting in increased early acrosome reaction and consequently decreasing their fertilizing capacity. Although the mechanisms involved in the regulation of sperm capacitation and acrosome reaction are not fully understood, it has been reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an important regulator of these processes. Thus, the present aimed to evaluate the effect of heat shock in the CaMKII signaling during the bovine sperm capacitation and acrosome. Methods:Bovine spermatozoa were in vitro capacitated for 4 hours. The acrosome reaction was induced by exposure to heparin and calcium ionophore A23187 for 1 hour. Heat shock was applied by incubating spermatozoa at 41 °C with 7% CO2, while the control group was maintained at 38.5 °C with 5% CO2. At the end of each treatment, the localization of total CaMKII and phosphorylated CaMKII (pCaMKII), as well as acrosomal membrane integrity, were evaluated by immunofluorescence. Results:It was observed that CaMKII and not phosphorylated CaMKII (pCaMKII) localization at the acrosome region was affected by sperm capacitation. In contrast, the localization of both, CaMKII and its phosphorylated form was affected by the acrosome reaction (p < 0.05). The acrosome membrane integrity, as well as the pCamKII localization in bovine spermatozoa, was affected by incubation time. This effect of incubation time was stronger in heated shock sperm, although it was observed only after 2 h of incubation. Heat shock also affected the acrosomal localization of pCaMKII in the acrosomal region of spermatozoa with intact acrosome. Discussion:Taken together, the data present here show that CaMKII and pCaMKII localization is dynamic during bovine sperm capacitation and acrosome reaction and that this pattern of localization is affected by heat shock, suggesting that failure in CaMKII signaling is probably involved in the early acrosome reaction observed in heated-shock spermatozoa.

Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41 degrees C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41 degrees C while mitochondrial activity was reduced by 38.5 and 41 degrees C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified > 300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41 degrees C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

In several species, oocyte and embryo competence are improved by the addition of endoplasmic reticulum (ER) stress inhibitors to in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. This study aimed to evaluate the effects of three concentrations of tauroursodeoxycholic acid (TUDCA; 50, 200, and 1,000 μM), a chemical chaperone for relieving ER stress, during IVM of bovine cumulus–oocyte complexes (COCs) for 24 h. Treated oocytes were analyzed for nuclear maturation, reactive oxygen species (ROS) production, mitochondrial activity, and abundance of target transcripts. In addition, the number of pronuclei in oocytes was evaluated after 18–20 h of insemination, and the rates of blastocyst and hatched blastocyst formation were evaluated after 7 and 8/9 days of culture, respectively. We further evaluated the transcript abundance of embryonic quality markers. Our findings showed that supplementation of IVM medium with 200 μM of TUDCA decreased ROS production and increased abundance of transcripts related to antioxidant activity in oocytes (CAT, GPX1, and HMOX1) and embryos (GPX1 and PRDX3). Interestingly, high concentration of TUDCA (1,000 μM) was toxic to oocytes, reducing the nuclear maturation rate, decreasing mitochondrial activity, and increasing the abundance of ER stress (HSPA5) and cellular apoptosis (CASP3 and CD40) related transcripts. The results of this study suggest that treatment with 200 μM of TUDCA is associated with a greater resistance to oxidative stress and indirectly with ER stress relief in bovine oocytes.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Porcine parvovirus (PPV) is one of the major infectious causes of reproductive failure of swine. This disease is characterized by embryonic and fetal infection and death, responsible for important economic losses. PPV is also implicated as a trigger in the development of post-weaning multisystemic wasting syndrome (PMWS) caused by Porcine circovirus type 2 (PCV2). Their detection is PCR-based, which is quite sensitive and specific, but laborious, costly and time-demanding. Therefore, this study aimed to assess Raman spectroscopy (RS) as a diagnostic tool for PPV and PCV2 due to its label-free properties and unique ability to search and identify molecular fingerprints. Briefly, swine testis (ST) cells were inoculated with PPV or PCV2 and in vitro cultured (37 degrees C, 5% CO2 ) for four days. Fixed cells were then submitted to RS investigation using a 633 nm laser. A total of 225 spectra centered at 1300 cm(-1) was obtained for each sample (5 spectra/cell; 15 cells/replicate; 3 replicates) of PPV-, PCV2-infected and uninfected (control) ST cells. Clear statistical discrimination between samples from both virus-infected cells was achieved with a Principal Component - Linear Discriminant Analysis (PCA-LDA) model, reaching sensitivity rates from 95.55% to 97.77%, respectively to PCV2- and PPV-infected cells. These results were then submitted to a Leave-One-Out (LOO) validation algorithm resulting in 99.97% of accuracy. Extensive band assignment was analyzed and compiled for better understanding of PPV and PCV2 virus-cell interaction, demonstrating that specific protein, lipids and DNA/RNA bands are the most important assignments related to discrimination of virus-infected from uninfected cells. In conclusion, these results represent promising bases for RS application on PCV2 and PPV detection for future diagnostic applications. (C) 2020 Elsevier B.V. All rights reserved.

Heat stress is an environmental factor that challenges livestock by disturbing animal homeostasis. Despite the broad detrimental effects of heat stress on reproductive function, the germline and the early preimplantation embryo are particularly prone. There is extensive evidence that elevated temperature reduces oocyte developmental competence through a series of cellular and molecular damages. Further research revealed that the oocyte respond to stress by activating cellular mechanisms such as heat shock response, unfolded protein response and autophagy to improve survival under heat shock. Such knowledge paved the way for the identification of thermoprotective molecules that alleviate heat-induced oocyte oxidative stress, organelle damage, and apoptosis. Therefore, this review depicts the deleterious effects of heat shock on oocyte developmental competence, heat-induced cellular and molecular changes, outlines pro-survival cellular mechanisms and explores thermoprotective molecules to improve oocyte competence.

Autophagy is a physiological mechanism that can be activated under stress conditions. However, the role of autophagy during oocyte maturation has been poorly investigated. Therefore, this study characterized the role of autophagy on developmental competence and gene expression of bovine oocytes exposed to heat shock (HS). Cumulus-oocyte-complexes (COCs) were matured at Control (38.5 °C) and HS (41 °C) temperatures in the presence of 0 and 10 mM 3-methyladenine (3MA; autophagy inhibitor). Western blotting analysis revealed that HS increased autophagy marker LC3-II/LC3-I ratio in oocytes. However, there was no effect of temperature for oocytes matured with 3MA. On cumulus cells, 3MA reduced LC3-II/LC3-I ratio regardless of temperature. Inhibition of autophagy during IVM of heat-shocked oocytes (3MA-41 °C) reduced cleavage and blastocyst rates compared to standard in vitro matured heat-shocked oocytes (IVM-41 °C). Therefore, the magnitude of HS detrimental effects was greater in the presence of autophagy inhibitor. Oocyte maturation under 3MA-41 °C reduced mRNA abundance for genes related to energy metabolism (MTIF3), heat shock response (HSF1), and oocyte maturation (HAS2 and GREM1). In conclusion, autophagy is a stress response induced on heat shocked oocytes. Inhibition of autophagy modulated key functional processes rendering the oocyte more susceptible to the deleterious effects of heat shock.

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 degrees C for 24 h (control group, CG), or incubated at 41 degrees C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 degrees C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 degrees C. These results provide evidence that HS during IVM for 12-22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.

Elevated temperature can compromise the ability of the mammalian oocyte to develop to the blastocyst stage after fertilization. The microenvironment of the oocyte is determined by the cellular and non-cellular components of the follicle including cumulus cells and follicular fluid. Here we tested whether follicular fluid contains molecules that can protect the bovine oocyte from heat shock during maturation, and if so, whether some of these protective molecules are present in exosomes. The experiments utilised ovaries from Bos taurus and admixtures of B. taurus and Bos indicus. Four separate pools of follicular fluid were prepared by aspiration of follicles from 48 to 70 slaughterhouse ovaries. Exosomes were isolated from follicular fluid by a series of centrifugation, filtration, and ultracentrifugation steps before being reconstituted in PBS. Each of the 4 exosome preparations was subject to particle size and concentration analysis. The experiments were designed as 2 × 3 factorial to test the effect of temperature and supplementation. Cumulus-oocyte complexes (COC) obtained from slaughterhouse ovaries were matured at 38.5°C for 22 h (control) or 41°C for 14 h followed by 38.5°C for 8 h (heat shock). Maturation was performed in the presence of vehicle (PBS), 10% (v/v) follicular fluid, or exosomes (16 × 109 particles/mL). Data were analysed by least-squares ANOVA. Orthogonal contrasts and the mean separation test pdiff were used to compare means. Effects of treatment on cumulus cell expansion (change in diameter after maturation) were replicated 5 times using 119 to 122 COC per treatment. Effects of treatment on embryonic development after fertilization of treated COC was determined in 6 replicates using 244 to 286 embryos per replicate. Expansion was reduced by heat shock (P < 0.001), and affected by treatment (P < 0.05), with both follicular fluid and exosomes preventing the decrease in expansion caused by heat shock. Cleavage was reduced by heat shock (P < 0.001) and affected by treatment (P < 0.05) and the interaction between temperature × supplementation (P < 0.05). Although heat shock reduced the cleavage rate for vehicle-treated oocytes (77 v. 67%), there was no effect of heat shock for oocytes treated with follicular fluid FF (78 v. 74%) or exosomes (79 v. 78%; SEM = 1.4%). Heat shock also reduced the percent of cleaved embryos becoming blastocysts for the vehicle group (27 v. 17%; P < 0.05) but had no effect on percent of cleaved embryos becoming blastocysts for the follicular fluid (31% v. 26%) or exosome groups (28 v. 26%). Uptake of exosomes into isolated cumulus cells and oocytes cultured at 38.5°C for 0.5, 1, 14 and 22 h was examined using labelling of exosomes with 10 µM BODIPY® Ceramide TR (Thermo Fisher Scieintific, Waltham, MA, USA) and confocal microscopy. Exosomes were taken up by cumulus cells after culture for 1 h or later but were not taken up by oocytes. In conclusion, follicular fluid exosomes protected the oocytes from heat shock and this effect seems to be mediated by cumulus cells. Study supported by BARD US-4719-14.

The modern definition of Epigenetics refers to heritable changes in gene function, and in turn in phenotypes, that are not caused by genetic alterations. It comprises DNA methylation, histone modifications, and noncoding RNAs that collectively compose the “epigenome” and orchestrates numerous essential regulatory mechanisms during mammalian development, including cell and tissue differentiation, genomic imprinting, and X chromosome inactivation. Unlike the genome, the epigenome is highly dynamic and is able to modulate genome function under exogenous influence. This remarkable ability accelerates adaptation and may be extremely beneficial; nevertheless, growing evidence indicates that epigenetic modifications exogenously induced around the time of conception or during early embryogenesis may be detrimental for the immediate and future health of the individual. The potential transmission of epigenetic variations to subsequent generations makes environmental exposure mostly critical. Not surprisingly, interest has recently grown on how exposure to environmental conditions (e.g., nutritional, hormonal, metabolic, or environmental variations) modulates establishment and maintenance of epigenetic modifications during early life and thereby affects gene expression and phenotype. The sensitivity of the early embryo to the environment was confirmed across animal models and in humans and associated with postnatal growth and metabolism. This periconceptional “programming” is in accordance with the Developmental Origins of Health and Disease (DOHaD) hypothesis, which proposes a profound influence of fetal environment on development, physiology, and risk of disease in adult life. This special issue presents a collection of research and review articles addressing cellular, molecular, and epigenetic changes in gametes or preimplantation embryos of different mammalian species following various environmental exposures. The topics include metabolic stress, hormone exposure, assisted reproductive technologies, diet, adverse temperature, and modified atmosphere conditions. The collection provides relevant insights on novel findings and will hopefully inspire new directions for future research. We would like to express our gratitude to all those who have made this Special Issue possible. We thank all the Authors for their excellent contributions and their patience as the Issue was being compiled, and the reviewers for their careful and altruistic work. We would also like to acknowledge Molecular Reproduction and Development Editorial Office for the excellent assistance. Finally, we express our great appreciation to Dr. Gary Wessel and Dr. Harvey Florman, former and current Editor in Chief of the journal for supporting our initiative.

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14 h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25 nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

Exposure of the preimplantation embryo to heat stress triggers a series of cellular, molecular, and adaptive changes preventing a normal embryonic development. Heat stress disrupts the embryo cytoskeleton, intracellular calcium levels, mitochondrial function, and induces apoptosis. Moreover, heat stress can act indirectly through induction of reactive oxygen species (ROS), leading to a variety of cellular damage. Embryonic resistance to heat shock is determined by factors such as genotype, developmental stage, apoptosis, redox status, and regulatory molecules. The early embryo is very susceptible to heat stress; it acquires resistance to elevated temperature as development advances. One of the mechanisms involved in the developmental acquisition of thermotolerance is heat-induced apoptosis, which acts as a quality control mechanism to remove damaged blastomeres allowing the embryo to survive after stress. Although embryos at >8-cell stage can activate the apoptotic cascade as an adaptive response to stress, embryos at the two-cell stage are resistant to proapoptotic signals. This lack of apoptotic response has been associated to mitochondrial resistance to depolarization and epigenetic regulations, such as DNA methylation and histone deacetylation. Even though the cellular mechanisms triggered by heat stress have been studied, very little attention has been paid to the vulnerability of the epigenome to drastic temperature changes during the preimplantation period. Therefore, this review aims to characterize the effects of elevated temperature on the bovine embryo, especially addresissing developmental, cellular, and epigenetic alterations triggered in response to temperature.

Diabetes mellitus type 2 (T2DM), characterized by insulin resistance and/or relative insulin production deficiency, has been shown to cause male infertility. Hyperglycemia is known to directly induce reactive oxygen species (ROS) production, resulting in oxidative stress. Bauhinia forficata Link (Bf L.) is a powerful source of natural antioxidants with hypoglycemic and antidiabetic activities. Due to these effects, Bf L has been suggested as an alternative treatment to minimize T2DM complications associated to oxidative stress. Therefore, the present study investigated Bf L effects on the spermatozoa function of T2DM mice. For that, 16 C57Bl/6J mice with streptozotocin (STZ)-high fat diet (HFD)-induced T2DM were randomly distributed on diabetes control group (T2DM-C) and diabetic group fed with aqueous extract of Bf L. fresh leaves decoction (Dec) (T2DM-B) for 6 weeks. After treatment, animals with glycaemia greater than 230 mg/dL were considered diabetic and epididymis cauda sperm recovered for analysis. The data was analyzed by t-test using MiniTab 17 software. Data are expressed as mean ± SEM. Epididymal sperm motility, as evaluated by CASA, was not affected by bauhinia treatment in any parameters analyzed. There was no effect of Bf L. on glutathione peroxidase (28.2 ± 5.1 and 25.3 ± 4.4 for T2DM-C and T2DM-B group, respectively) and superoxide dismutase (59.8 ± 18.3 and 55.2 ± 11.8 for T2DM-C and T2DM-B group, respectively) sperm activity. Lipid peroxidation as assessed by thiobarbituric acid reactive substances (TBARS) was not different between T2DM-C (163.6 ± 29.2) and T2DM-B (137.3 ± 28.8) mice sperm cells. ROS evaluation by flow cytometry using CelRox probe showed that ROS positive sperm in T2DM-C (4.40 ± 1.9) was not different from those observed in T2DM-B mice sperm (9.80 ± 2.9). Similarly, sperm high mitochondrial potential evaluated by flow cytometry using JC-1 probe was similar between T2DM-C (10.86 ± 3.4) and T2DM-B (19.88 ± 3.7) mice. In conclusion, Bauhinia forficata Link did not affect function and redox status of spermatozoa from STZHDF-induced diabetic mouse. Recipient of the “Young Talent Attraction" Fellowship from Science without border CAPES - CSF-PAJT - 88887.068701/2014-00.

Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100ng mL-1) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5°C (control) or 41°C (heat shock; HS) for 14h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41°C increased (P<0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25ng mL-1, IGF1 tended to minimise these negative effect of HS (P=0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P<0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P<0.05) by 12.5ng mL-1 IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 °C for 22 hours) and heat shock (41 °C for 14 hours followed by 38.5 °C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 °C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.

Follicular fluid (FF) is a component of oocyte microenvironment containing plasma factors and specialized molecules secreted by follicular cells and oocyte. Follicular fluid products play an important role in follicular growth and oocyte maturation. Heat stress has been shown to compromise the follicular microenvironment and oocyte maturation. Exposure of bovine oocytes to elevated temperature compromise oocyte maturation and developmental competence. Therefore, the objective of this study was to determine the role of FF on oocyte nuclear maturation and cumulus cells (CCs) expansion in cumulus-oocyte complexes (COCs) subjected to heat shocked. Follicular fluid was collected by aspiration from slaughterhouse ovaries during the winter months, processed and stored at -80oC. Slaughterhouse COCs were matured in Maturation Medium (MM) (TCM199-Bicarbonate with 50 μg/mL gentamicin, 0.2 mM sodium pyruvate, 10 μg/mL FSH, 10 μg/mL LH and 1 μg/mL estradiol 17-b). COCs were distributed in positive control (MM + 10% fetal bovine serum at 38.5oC for 22 h), 0% FF control (MM + 0% FF at 38.5oC for 22 h) and heat shock (MM at 41oC for 14 h followed by 38.5°C for 8 h) in the presence of 0, 10, 15 e 20% FF during in vitro maturation (IVM). After 22h IVM, CCs expansion was evaluated by image analysis (software ImageJ) of each COC before and after IVM (N = 5 replicates using 139-154 COCs/treatment). COCs were vortexed in 100 mg/mL hyaluronidase for 5 minutes to remove cumulus cells. Denuded oocytes were fixed in 3.7% formaldehyde for 30 minutes and stained with 1 mM Hoechst 33342 for 15 minutes to determine meiotic progression (N = 6 replicates using 126–136 COCs/treatment). Data were analyzed by ANOVA (SAS). Heat shock reduced CCs expansion (P < 0.001) from 3.38 ± 0.14 (positive control) and 3.00 ± 0.14 (0%FF control) to 1.91 ± 0.14 fold (0% FF heat shock). The proportion of metaphase II (MII) oocytes was reduced (P < 0.001) by heat shock from 84.0 ± 4.0% in positive control and 74.9 ± 4.0% in 0% FF control to 46.5 ± 4.0% in 0% FF heat shock. However, addition of 10.15 and 20% FF rescued the deleterious effect of heat shock in CCs expansion (2.62 ± 0.14, 2.63 ± 0.14 and 2.63 ± 0.14 fold for 10.15 and 20% FF, respectively) while the doses of 10 and 15% FF rescued nuclear maturation of heat shocked oocytes (64.5 ± 4.0% and 64.0 ± 4% for 10 and 15% FF, respectively) which was similar to 0% FF control at 38.5°C. In conclusion, addition of 10 and 15% FF to MM rescued oocyte expansion and nuclear maturation of heat shocked bovine oocytes, suggesting that FF factors prevent the deleterious effect of heat shock.

Bull heat stress increased testicular temperature reducing sperm production, motility and increasing the percentage of abnormal sperm morphology. Establishment of an in vitro heat stress model for bull sperm requires medium validation to evaluate cellular function based on sperm motility and mitochondrial activity. SP-TALP (Tyrodealbumin-lactate-piruvate) and TL-semen chemical composition are similar, except for BSA (Bovine Serum Albumin), lactate and piruvate present on SP-TALP (Bavister, Biol Reprod, 16:228-237,1977). Therefore, the objective of this study was to establish an in vitro heat stress model for Bos taurus taurus sperm evaluating motility and mitochondrial activity. Frozen semen straws (n=30) from four Holstein bulls were used (N= pool of 3 straws/replicate). Samples were evaluated immediately after Pecoll Gradient (0 hour) and after SP-TALP and TLsemen incubation at 35°C, 38.5°C and 41°C during 4 hours. Sperm motility was determined at 0, 1, 2, 3 and 4 hour incubation and mitochondrial activity (Hoechst 33342/MitotrackerRed) at 0 and 4 hour incubation. Data were submitted to ANOVA and non parametric data were analyzed by Wilcoxon using SAS 9.0. SP-TALP sperm incubation at 35°C (54 ± 4.07%); 38.5°C (53 ± 4.07%) and 41°C (47 ± 4.07%) for 2 hours did not affect motility as compared to 0 h (64 ± 4.07%). However, SP-TALP sperm incubation at 41°C for 3 hours reduced (34 ± 4.07; P < 0.02) sperm motility as compared to 0 h (64 ± 4.07%). TL-semen sperm incubation at 41°C for 1 h reduced motility (22 ± 3.87%; P < 0.05) as compared to 35°C (43 ± 3.87%; P < 0.05) and control 0 h (46 ± 3.87%). Incubation of sperm at 38.5°C (1.42 ± 0.017 arbitrary units (AU), P = 0.05) and at 41°C (1.41 ± 0,017 UA; P < 0.05) for 4 hours reduced mitochondrial activity as compared to 35°C (1.48 ± 0.017 AU; P < 0.05) regardless of medium. Mitochondrial activity of sperm incubated in SP-TALP medium (1.44 ± 0.014 AU) was superior (P < 0.05) than TLsemen (1.43 ± 0.014 AU) regardless of temperature. These observations suggested that important medium compounds for sperm function are lost in sperm incubated in TL-semen, but it was maintained if incorporated in SPTALP. In conclusion, in vitro heat stress model using SP-TALP medium provided enriched energy substrate for sperm motility and mitochondrial activity