Sperm quality influences bovine in vitro embryo production (IVEP). Linear regression is a statistical tool that models the relationship between a dependent variable and one or more independent variables. It can be used to predict outcomes, analyze trends, and understand the impact of variables. These models are useful for indicating which sperm variables most influence IVEP results, facilitating the selection of superior samples to enhance IVEP. Using early IVEP indicators, such as cleavage rate, can assist in scheduling recipient preparation. This work aimed to construct linear regression models to study the influence of a comprehensive set of sperm variables and cleavage rate on IVEP yields. A dataset comprising 51 semen batches from 23 Nellore bulls was compiled, including 26 sperm variables from computer-assisted sperm analysis (CASA) and flow cytometry per batch, with 184 IVEP procedures. The most robust predictive model had a coefficient of determination of 0.6358; furthermore, the BULL variable was the most influential predictor, yielding an independent coefficient of determination of 0.5218. Models that were exclusively founded on sperm analysis yielded meager coefficients of determination (<0.04). However, to predict the best batch from a bull, individual models achieve coefficients of determination ranging from 0.58 to 0.99. Contributions, impacts, and positive or negative correlations of various sperm variables with in vitro performance were influenced by the bull. We conclude that the BULL variable was the dominant predictor of in vitro performance, with cleavage rates serving as an early estimator of blastocyst rates. The predictive utility of analyzed sperm traits remains limited. Nonetheless, individualized models offer a valuable tool for selecting optimal batches for preferred bulls within IVEP laboratories, culminating in heightened blastocyst rates.

The formation of the blastocyst is a highly orchestrated process involving different cellular and molecular aspects. In mice, the mTOR pathway influences the Hippo pathway, leading to expression of trophectoderm (TE)-related genes. However, these mechanisms are not fully characterized in bovine embryos, and biological pathways are not always conserved across species. We hypothesized that mTOR pathway positively influences the TE differentiation in bovine embryos. This study aimed to evaluate the effects of the mTOR agonist MHY1485 (MHY) on TE formation in in vitro-produced bovine embryos. Embryos were treated at 96 h post insemination (hpi) with 2 µM MHY or DMSO (vehicle) until 144 hpi or 192hpi. At 144 hpi, morulae were collected for gene expression analysis of CDX2, GATA3, OCT4, SOX2, TFAP2C and YAP1. No significant differences in mRNA expression were observed between the Control, DMSO or MHY groups. At 192 hpi, embryos were fixed for confocal microscopy to assess the TE marker GATA3 and the Hippo-related transcription factor YAP1. While total cell and inner cell mass counts remained unaffected, TE cell numbers were significantly reduced in the DMSO group compared to both the Control and MHY groups. Furthermore, nuclear YAP1 intensity was lower in the DMSO group compared to the MHY group, in which YAP1 was lower than the Control. In conclusion, pharmacological activation of the mTOR pathway did not positively influence TE differentiation in bovine embryos; however, it effectively rescued the unexpected negative effects induced by the vehicle (DMSO) on TE cell number and YAP1 signaling.

Research on reproductive advanced age has predominantly focused on women due to their biological clock. The impact of paternal senescence on fertility and offspring is still poorly understood. In this study, we characterized the effects of aging on sexual behavior and reproductive characteristics using male mice aged 4 months as a control, compared to mice aged 19 and 24 months. In the first experiment, we observed that reproductive interest and locomotor activity were reduced in older males, accompanied by reduced serum testosterone levels and testicular weight. We observed increased sperm morphological anomalies, protamine deficiency, and reduced sperm capacitation in aging mice. Aging also influenced cleavage, blastocyst and in vitro embryo development rates; embryo kinetics, and initial cell differentiation. In the second experiment, aging promoted smaller, lighter fetuses and a lower fetal weight ratio. In conclusion, an increased age impaired sexual behavior, sperm function, and embryo and fetal development.

Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), could be an alternative to improve cryopreserved canine sperm by increasing membrane fluidity. However, PUFAs also increase sperm susceptibility to lipid peroxidation. Supplementation of the extender with DHA and antioxidants could be an alternative. We evaluated supplementation of antioxidants (vitamin E and catalase) of cryopreserved canine semen extended with DHA. In Experiment 1, ejaculates from 8 dogs were divided into 4 aliquots, with increasing concentrations of DHA in the extender (0, 1, 5, and 10 µM) and cryopreserved. Sperm motility increased in 5 µM DHA and it was used for Experiment 2; ejaculates from 8 dogs were divided into 4 groups: control (5 µM DHA), vitamin E + DHA, catalase + DHA, and vitamin E + catalase + DHA. Samples were evaluated for susceptibility to lipid peroxidation, sperm kinetics, mitochondrial function, DNA integrity, and plasma and acrosomal membrane integrity. Vitamin E + DHA group had beneficial effects on the characteristics of sperm kinetics whereas the catalase + DHA group was harmful to sperm mitochondria. Vitamin E + catalase + DHA group decreased oxidative stress and damage to the plasma membrane and had low mitochondrial membrane activity. Addition of DHA + vitamin E in canine semen extender improved sperm quality after cryopreservation.

Methodologies used to evaluate bull fertility often overlook sperm chromatin, a nuclear structure critical for reproductive success due to its DNA composition, which is tightly bound to nucleoproteins called protamines. In mammals, the ratio between Protamine 1 (PRM1) and Protamine 2 (PRM2) plays a pivotal role in male fertility, with imbalances linked to infertility in species such as humans and mice. While bull sperm chromatin was previously believed to contain only PRM1, recent findings have confirmed the presence of PRM2, prompting questions about the impact of the PRM1:PRM2 ratio on bull fertility. The present study hypothesizes that bull infertility may be associated with impaired protamination, resulting in imbalances between PRM1 and PRM2 gene expression. The objective was to compare the chromatin structure through protamine deficiency (CMA3), DNA susceptibility to fragmentation, and PRM1 and PRM2 gene expression among bulls of differing in vivo fertility levels (high fertility and low fertility) and breeds (Nellore and Angus). Sperm from 14 Nellore and 20 Angus bulls with high and low in vivo fertility were analyzed for protamine deficiency, susceptibility to DNA fragmentation, and PRM1 and PRM2 gene expression. Additionally, PRM1:PRM2 ratios were calculated for each experimental group. Results revealed solely a breed effect. Nellore sperm exhibited lower susceptibility to DNA fragmentation and higher PRM1 and PRM2 total transcripts compared to Angus. Conversely, Nellore sperm also demonstrated greater susceptibility to protamine deficiency. Protamine composition may not explain fertility differences, it could play a significant role in adaptive mechanisms across bull breeds.

The aim of the present study was to improve bioenergetics and oxidative status of cryopreserved ram sperm by uncoupling mitochondrial activity and stimulating glycolysis. To verify a potential synergism between mitochondrial uncoupling and glycolysis stimulation, as well as to determine the optimal concentrations of the respective treatments, the study was divided into two experiments. In Experiment 1, ejaculates from eight rams were diluted with the commercial extender (Botubov®), supplemented with the mitochondrial uncoupler CCCP (0, 1, 10, and 20 μM), with or without 5 mM glucose, and then subjected to cryopreservation. After thawing, sperm function and oxidative status analyses were conducted to determine the optimal CCCP concentration, which was selected for Experiment 2. In Experiment 2, ejaculates from seven rams were diluted with the commercial extender (Botubov®) and supplemented with CCCP at doses of 0, 2.5, 5, and 10 μM, with or without 5 mM glucose. After thawing, an analysis of sperm bioenergetics was performed. Differences between treatments were assessed using ANOVA, followed by LSD mean comparison test for the combination of factors. In both experiments, total and progressive motility were higher in the CCCP 10 μM + glucose 5 mM group. This same group exhibited less susceptibility to lipid peroxidation, lower DNA fragmentation (Experiment 1), and greater mitochondrial activity (Experiment 2). Furthermore, treatments with only CCCP were deleterious to sperm. In conclusion, the use of the mitochondrial uncoupler CCCP at a dose of 10 μM combined with 5 mM glucose was promising in improving post-thaw sperm attributes.

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.

The study aimed to compare the impact of liquid nitrogen (LN2) levels within the storage dewar on sperm quality and investigate the effects of the nitrogen level on the sperm straws stored at the top or bottom of the semen racks. A total of 48 straws from 8 different bulls were used. Analyses were performed at three different LN2 levels within the dewar (30, 15, and 7 cm), and at each level, two straws (one from the top and one from the bottom of the rack) from each bull were used. Analyses included assessing mitochondrial membrane potential (MMP), plasma and acrosomal membrane integrity (PMAI), and sperm kinematics using CASA. Neither the evaluated nitrogen levels nor the straw storage location in the top or bottom goblet of the rack affected sperm quality. There were no differences in total and progressive motility or MMP and PMAI. The results obtained in this study show that, as long as the internal temperature in the dewar is maintained at -194 °C or below, even at the lowest evaluated liquid nitrogen level, there is no loss of sperm quality, regardless of the storage location of the straw in the rack.

Efficient spermatogenesis in mammals occurs when testicular temperature is approximately 2 to 8 °C below body temperature. Elevated testicular temperature can trigger oxidative stress and compromise sperm integrity during spermatogenesis, potentially resulting in damaged spermatozoa and male infertility. This study aimed to evaluate how heat stress affects the quantity of seminiferous tubules, and the abundance of germ cells within the seminiferous tubules. To this end, six Santa Inês rams were subjected to testicular insulation for 12 consecutive days, followed by two hemi-orchiectomies, the first 24 hours after insulation period to evaluate the immediate effect, and the second 30 days after the first hemi-orchiectomy to evaluate the late effect. Six Santa Inês rams composed the control group. Histological analyses were conducted to quantify the number of seminiferous tubules and the types of cells within them (spermatogonia, spermatocytes, and spermatids) in testicular fragments. Despite an increase in testicular temperature, no significant differences were observed in the number of seminiferous tubules. These findings probably reflect the resistance of Santa Ines rams to high environment temperatures. Regarding the abundance of cells, a decrease in spermatogonia (0.27% ± 0.06; 0.05% ± 0.03, p = 0.005) and an increase in spermatocytes (35.90% ± 1.58; 46.77% ± 4.33, p = 0.028) were observed immediately after the insulation period compared to 30 days after, the late effect. This result suggests an effect of the first hemi-orchiectomy on the remaining testicle, probably an attempt to maintain sperm production.

Male fertility potential depends on physical, endocrine, and genetic factors responsible for producing functional male gametes. Although the main function of the male gamete, the spermatozoon, is to deliver its genetic material to the oocyte, this premise has been modified over the past few years. It is believed that the spermatozoon provides essential factors for fertilization and pre-implantation embryo development. A viable/healthy spermatozoon has functional subcellular compartments (nucleus, acrosome, midpiece, and flagellum) due to the actions of proteins, transcripts, and epigenetic marks in the organelles present in them that have important roles in reproductive biology. Male fertility potential reflects viable spermatozoa with proper function. Therefore, new approaches to functional sperm analysis are essential. Additionally, intrinsic factors and sperm molecules constitute potential biomarkers of viable spermatozoa and male fertility. Among these factors are proteins, the genome, and coding and non-coding RNAs, such as microRNAs, that act during fertilization and early embryo development. Research has been seeking increasingly efficient tools to predict fertility and functional studies of these molecules through gene and protein expression. Thus, analytical tools are essential to identify and classify viable and functional spermatozoa, to evaluate assisted reproductive male potential.

There are more than 200 species and subspecies of Neotropical Primates of which more than 40

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen - CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and alpha-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mecha-nisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 x 106 cells/ mL) were incubated for 1 h at 38.5 degrees C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p < 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to in-fluence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to in-crease SMGT protocols, without altering the sperm viability.

The use of different sires influences in vitro embryo production (IVP) outcome. Paternal effects are observed from the first cleavages until after embryonic genome activation (EGA). Little is known about the mechanisms that promote in vitro fertility differences, even less about the consequences on embryo development. Therefore, this study aimed to evaluate the paternal effect at fertilization, embryo developmental kinetics, gene expression and quality from high and low in vitro fertility bulls. A retrospective analysis for bull selection was performed using the In vitro Brazil company database from 2012 to 2015. The dataset was edited employing cleavage and blastocyst rates ranking a total of 140 bulls. Subsequently, the dataset was restricted by embryo development rate (blastocyst/cleaved rate) and ten bulls were selected as high (HF; n = 5) and low (LF; n = 5) in vitro fertility groups. IVP embryos derived from high and low fertility bulls were classified according to their stage of development (2 cells, 3-4 cells, 6 cells, 8-16 cells), at 24, 36, 48, 60, 72 hpi, respectively, to evaluate embryo kinetics. Pronuclei formation (24 hpi), cleavage rate (Day 3), development rate, and blastocyst morphology (Grade I and II - Day 7) were also assessed, as well as the abundance of 96 transcripts at 8-16 cell stage and blastocysts. There was no difference in early embryo kinetics (P > 0.05), and cleavage rate (HF = 86.7%; LF = 84.9%; P = 0.25). Nevertheless, the fertilization rate was higher on HF (72%) than LF (62%) and the polyspermy rate was lower on HF compared to LF (HF:16.2% LF:29.2%). As expected, blastocyst rate (HF = 29.4%; LF = 16.0%; P < 0.0001) and development rate (HF = 33.9% LF = 18.9%; P < 0.0001) were higher in HF than LF. At the 8-16 cell stage, 22 transcripts were differentially represented (P <= 0.05) between the two groups. Only PGK1 and TFAM levels were higher in HF while transcripts related to stress (6/22, similar to 27%), cell proliferation (6/22, similar to 27%), lipid metabolism genes (5/22, similar to 23%), and other cellular functions (5/22, similar to 23%) were higher on LF embryos. Blastocysts had 9 differentially represented transcripts (P <= 0.05); being only ACSL3 and ELOV1 higher in the HF group. Lipid metabolism genes (3/9, 33%) and other cellular functions (6/9, 67%) were higher in the LF group. In conclusion, the timing of the first cleavages is not affected by in vitro bull fertility. However, low in vitro fertility bulls presented higher polyspermy rates and produced 8-16 cells embryos with higher levels of transcripts related to apoptosis and cell damage pathways compared to high in vitro fertility ones. Evidence such as polyspermy and increase in apoptotic and oxidative stress genes at the EGA stage suggest that embryo development is impaired in the LF group leading to the reduction of blastocyst rate. (C)2021 Elsevier Inc. All rights reserved.

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

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

Sperm routinary fitness evaluation is not sufficient to predict bull reproductive capacity as they present differences in fertility up to 40%. Among the defects which compromise spermatozoa functionality, new approaches consider the study of sperm chromatin, which is the core structure containing paternal genetic information. Sperm chromatin needs to be compacted to maintain the integrity of DNA, which occurs by binding nucleoproteins with high affinity to DNA. In the last stages of sperm maturation, chromatin is hyper-compacted by basic proteins called protamines in a process named protamination. In this review, we summarized intrinsic and extrinsic factors that are suggested to influence protamination in bull spermatozoa, considering old and new evidence from human and murine spermatozoa. Also, the current approaches to evaluate bull protamination and its relationship with fertility were described. Nevertheless, the physiological mechanisms of protamination are still poorly understood.