Female Reproductive Fluids Research Articles

Sperm performance in the race for fertilization, the influence of female reproductive fluid.

In studies of sperm competition, particularly in external fertilizers, the importance of the fertilization environment on the paternity share among rival males often goes overlooked. The female reproductive fluid (FRF), produced and released by females, creates the microenvironment that sperm encounter on their quest for fertilization and can generate paternity biases by affecting key traits in sperm competition. Yet, whether there is a direct link between FRF effects on sperm traits and its effect on competitive fertilization dynamics remains to be explored. Here, using the zebrafish Danio rerio, we compare within-female paternity share among two competing males and predictors of fertilization success (i.e. sperm traits) in the presence/absence of FRF. Our results unequivocally reveal a direct link between the direction and magnitude of the effect of FRF on sperm traits and the change in the competitive fertilization success of each male. This study demonstrates that the FRF directly mediates post-mating female control through its differential effect on sperm performance and that the FRF's effect on sperm quality alone is sufficient to predict the magnitude of the fitness effects. These findings highlight the need to consider the role of FRF in fertilization, avoiding biases resulting from an exclusive focus on male intrinsic sperm quality.

Proteomic analysis reveals dynamic changes in cloacal fluid composition during the reproductive season in a sexually promiscuous passerine

Cryptic female choice (CFC) is a component of postcopulatory sexual selection that allows females to influence the fertilization success of sperm from different males. While its precise mechanisms remain unclear, they may involve the influence of the protein composition of the female reproductive fluids on sperm functionality. This study maps the protein composition of the cloacal fluid across different phases of female reproductive cycle in a sexually promiscuous passerine, the barn swallow. Similar to mammals, the protein composition in the female reproductive tract differed between receptive (when females copulate) and nonreceptive phases. With the change in the protein background, the enriched gene ontology terms also shifted. Within the receptive phase, distinctions were observed between proteomes sampled just before and during egg laying. However, three proteins exhibited increased abundance during the entire receptive phase compared to nonreceptive phases. These proteins are candidates in cryptic female choice, as all of them can influence the functionality of sperm or sperm-egg interaction. Our study demonstrates dynamic changes in the cloacal environment throughout the avian breeding cycle, emphasizing the importance of considering these fluctuations in studies of cryptic female choice.

A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

A ketogenic diet (KD) elevates blood β-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. A ketogenic diet elevates blood β-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters β-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and β-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and β-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated β-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Paternal environment effects are driven by female reproductive fluid but not sperm age in an external fertilizer.

Sperm ageing after ejaculation can generate paternal environment effects that impact offspring fitness. In many species, female reproductive fluids (FRFs), i.e. ancillary fluids released by eggs or within the female reproductive tract, may protect sperm from ageing and can additionally interact with sperm to influence offspring viability. This raises the intriguing prospect that FRFs may alleviate paternal effects associated with sperm ageing. Here, we test this novel hypothesis using the broadcast spawning mussel, Mytilus galloprovincialis. We show that incubating sperm in FRF prior to fertilization increases offspring viability, and that these effects occur independently of sperm age. Our results provide novel evidence that FRFs allow females to selectively bias fertilization toward higher quality sperm within an ejaculate, which in turn yields more viable offspring. We consider this FRF-mediated paternal effect in the context of female physiological control over fertilization and the transgenerational effects of female-regulated haploid selection.

Effects of Benzo[a]pyrene on Human Sperm Functions: An In Vitro Study.

Benzo(a)pyrene (BaP) is considered one of the most dangerous air pollutants for adverse health effects, including reproductive toxicity. It is found both in male and female reproductive fluids likely affecting spermatozoa after the selection process through cervical mucus, a process mimicked in vitro with the swim-up procedure. In vitro effects of BaP (1, 5, 10 µM) were evaluated both in unselected and swim-up selected spermatozoa after 3 and 24 h of incubation. BaP reduced total, progressive and hyperactivated motility and migration in a viscous medium both in swim-up selected and unselected spermatozoa. Viability was not significantly affected in swim-up selected but was reduced in unselected spermatozoa. In swim-up selected spermatozoa, increases in the percentage of spontaneous acrosome reaction and DNA fragmentation were observed after 24 h of incubation, whereas no differences between the control and BaP-treated samples were observed in caspase-3 and -7 activity, indicating no effects on apoptotic pathways. ROS species, evaluated by staining with CellROX® Orange and Dihydroethidium, did not differ in viable spermatozoa after BaP treatment. Conversely, the percentage of unviable ROS-positive spermatozoa increased. Our study suggests that BaP present in male and female genital fluids may heavily affect reproductive functions of human spermatozoa.

Female reproductive fluid and male seminal fluid: a non-gametic conflict for post-mating control.

Growing evidence shows that non-gametic components released by both males and females can significantly drive sperm competition outcomes. Seminal fluid (SF) was shown to influence paternity success by affecting rival males' sperm performance, and, in some species with male alternative reproductive tactics, to selectively decrease the fertilization success of males of the opposite tactic. Female reproductive fluid (FRF) has been proven to differentially influence ejaculates of different males and bias fertilization towards specific partners. Whether, and with what outcome, these two processes can intersect to influence sperm competition is still unknown. Here we explore this scenario in the grass goby (Zosterisessor ophiocephalus), a fish with territorial-sneaker reproductive tactics, where sneaker males can exploit the territorials' SF while penalizing territorial sperm performance with their own fluid. To test whether FRF can rebalance the ejaculate competition in favour of territorial males, we used in vitro fertilization with a SF mixture (territorial + sneaker), using increasing concentrations of FRF, to simulate the natural conditions that ejaculates encounter towards the eggs. Our findings revealed a differential effect of FRF on the different tactics' fertilization success, favouring territorial ejaculates, possibly through an attenuation of the detrimental effects of sneaker SF, and enabling females to regain control over the fertilization process.

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer.

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.

Female reproductive fluid attracts more and better sperm: implications for within-ejaculate cryptic female choice.

Mounting evidence shows that the female reproductive fluid (FRF) can differently affect sperm performance of different males by biasing paternity share among competing males. Here, we tested for the first time the potential of 'within-ejaculate cryptic female choice' mediated by the FRF in the zebrafish (Danio rerio). Using a recently developed sperm selection chamber, we separated and collected FRF-selected from non-selected sperm to compare the two subpopulations of sperm in terms of sperm number, viability, DNA integrity and fertilizing ability. We showed that the sperm attracted by FRF are more numerous, more viable and with higher DNA integrity. In addition, FRF-selected sperm fertilized more eggs, but if this is due to fertilization ability per se or numerical advantage remains to be tested. Our results suggest that FRF can select sperm with a better phenotype, highlighting the crucial and impactful role that FRF might play in the process of fertilization and post-mating sexual selection dynamics, along with the potential implications for sperm selection in assisted reproductive techniques.

Female reproductive fluids 'rescue' sperm from phenotypic ageing in an external fertilizer.

Female reproductive fluids (FRFs) serve key reproductive functions in sexually reproducing animals, including modifying the way sperm swim and detect eggs, and influencing sperm lifespan. Despite the central role of FRF during fertilization, we know surprisingly little about sperm-FRF interactions under different environmental conditions. Theory suggests that in external fertilizers FRF may 'rescue' sperm from ageing effects as they search to fertilize eggs. Here, we test the interaction between these two fundamental properties of the fertilization environment, ejaculate age (i.e. time since ejaculation) and FRF, on a range of functional sperm phenotypes in a broadcast spawning mussel, Mytilus galloprovincialis. We found that the effects of ejaculate age on multivariate sperm motility traits and total sperm motility were altered by FRF, and that longer-lived sperm exhibit stronger, likely more advantageous, responses to FRF after periods of ageing. We also detected significant among-male variation in the relationship between sperm motility traits and ejaculate age; notably, these patterns were only revealed when sperm encountered FRF. Collectively these findings underscore the importance of considering female reproductive physiology when interpreting ageing-related declines in sperm motility, as doing so may expose importance sources of variation in sperm phenotypic plasticity among males and environments.

Examining the potential for resource-dependent female reproductive fluid-sperm interactive effects in a livebearing fish.

Sexually selected traits can be costly to produce and maintain. The amount of resources available to an individual is therefore expected to influence investment in costly sexual traits. While resource-dependent expression of sexually selected traits has traditionally been examined in males, resource limitation can also influence how sexual selection operates in females. Female reproductive fluids are thought to be costly to produce and may play an important role in shaping the outcome of postcopulatory sexual selection by influencing sperm performance. However, we know surprisingly little about whether and how female reproductive fluids are influenced by resource limitation. Here, we examine if resource restriction influences female reproductive fluid-sperm interactive effects in the pygmy halfbeak (Dermogenys collettei), a small internally fertilizing freshwater fish where females store sperm. After experimentally altering female diets (high vs. restricted diets), we compared how female reproductive fluids influence two key metrics of sperm quality: sperm viability and velocity. While female reproductive fluids enhanced sperm viability and velocity, we found no evidence that female diet influenced the interactive effect between female reproductive fluids and sperm viability or velocity. Our findings build on the growing evidence that female reproductive fluids influence sperm performance and call for further attention to be devoted to understanding how resource quantity and quality influence how female reproductive fluids affect sperm performance.

Female Reproductive Fluid Increases the Opportunities for Postmating Sexual Selection by Prolonging the Egg Fertilization Window.

AbstractFemale reproductive fluid (the fluid that surrounds the eggs) has attracted increasing attention for its role in fertilization and postmating sexual selection through its effects on sperm traits. Surprisingly, however, only a few studies have investigated the effects of the female reproductive fluid on the eggs. Yet these effects might offer great potential to affect fertilization dynamics by, for example, increasing the opportunities for postmating sexual selection. Here, we determined whether the female reproductive fluid, by extending the egg fertilization window (the time available for egg fertilization), could also increase the opportunities for multiple paternity. Using the zebrafish (Danio rerio), we first tested the prediction that female reproductive fluid increases the egg fertilization window; then, using a split-brood design with the sperm of two males added at different time points after egg activation, we tested whether the degree of multiple paternity varies in the presence or absence of female reproductive fluid. Our results reveal the potential of female reproductive fluid to increase multiple paternity through its effects on the egg fertilization window, thus broadening our knowledge of how female mechanisms affect postmating sexual selection in externally fertilizing species.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

Sperm Accumulation Induced by the Female Reproductive Fluid: Putative Evidence of Chemoattraction Using a New Tool.

There is considerable evidence that female reproductive fluid (FRF) interacts intimately with sperm, affecting several sperm traits, including sperm motility and longevity, and ultimately fertilization success. One of the first documented interactions between FRF and sperm is the ability of FRF to attract and guide sperm towards the eggs. However, most of the evidence of FRF’s chemoattraction proprieties comes from a limited number of taxa, specifically mammals and invertebrate broadcasting spawners. In other species, small FRF volumes and/or short sperm longevity often impose methodological difficulties resulting in this gap in chemoattraction studies in non-model species. One of the outcomes of sperm chemotaxis is sperm accumulation towards high chemoattractant concentrations, which can be easily quantified by measuring sperm concentration. Here, we tested sperm accumulation towards FRF in the zebrafish, Danio rerio, using an ad hoc developed, 3D printed, device (‘sperm selection chamber’). This easy-to-use tool allows to select and collect the sperm that swim towards a chemical gradient, and accumulate in a chemoattractant-filled well thus providing putative evidence for chemoattraction. We found that sperm accumulate in FRF in zebrafish. We also found that none of the sperm quality traits we measured (sperm swimming velocity and trajectory, sperm motility, and longevity) were correlated with this response. Together with the 3D printable project, we provide a detailed protocol for using the selection chamber. The chamber is optimized for the zebrafish, but it can be easily adapted for other species. Our device lays the foundation for a standardized way to measure sperm accumulation and in general chemoattraction, stimulating future research aimed at understanding the role and the mechanisms of sperm chemoattraction by FRF.

Antioxidants Present in Reproductive Tract Fluids and Their Relevance for Fertility.

Nowadays, infertility is classified as a disease of the reproductive system. Although it does not compromise the life of the individual, it can have detrimental effects on the physiological and psychological health of the couple. Male fertility evaluation is mainly focused on the analysis of sperm parameters. However, the ejaculated fluid is also composed of seminal plasma, and the study of this fluid can provide crucial information to help in the assessment of male fertility status. Total antioxidant capacity of the seminal plasma has been positively correlated with the fertility of men. Moreover, evidence highlights to a similar importance as that of female reproductive tract fluid antioxidant capabilities and female fertility. Herein, we describe the functions of seminal plasma and female reproductive tract fluids, as well as their main antioxidant components and their relationships with fertility outcomes. Additionally, this review contains the most up to date information regarding the mechanisms of the interaction between the male and the female reproductive fluids and the importance of proper antioxidant capacity for fertilization.

Physicochemical and Functional Characterization of Female Reproductive Fluids: A Report of the First Two Infants Born Following Addition of Their Mother's Fluids to the Embryo Culture Media.

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

Less is more: Reduced nutrient concentration during in vitro culture improves embryo production rates and morphophysiology of bovine embryos

Reproducing the environment to which the embryo is naturally exposed may be an alternative to improve viability of embryos produced in vitro. In the first part of this work, we describe a novel culture media, namely Embryonic Culture Supplementation (ECS100). The composition of this media was based on the contents of carbohydrates and amino acids found in oviductal and uterine fluids. Because it was a new formulation, we investigated the performance of ECS100 in comparison with conventionally used SOFaa, and possible benefits to embryo development. Embryo production rates (cleavage, morula and blastocyst conversion, blastocyst and hatching rates) and morphophysiological parameters (total cell number, cell allocation, Mitochondrial membrane potential (MMP), Reactive Oxygen Species (ROS), NADH, FAD+ and ATP content) were similar between ECS100 and SOFaa. Next, we tested if a reduction of ECS100 concentration could positively contribute to embryo viability by resembling the more dynamic availability of nutrients that reach the embryos in vivo. Therefore, embryos were cultured in ECS100 or in its serial dilution (ECS75, 50 and 25). Despite the fact that the lowest concentration (ECS25) still supported blastocyst formation, halving the concentration of metabolites (ECS50) actually improved embryo production rates. Thus, embryos produced in ECS100 or ECS50 were submitted to further analyses on Days 4 and 7. Embryos cultured in ECS50 presented better developmental rates and morphophysiological profile than embryos cultured in ECS100. Additionally, physiological traits (MMP, ROS and NADH levels) of embryos cultured in ECS50 presented the expected pattern for embryos produced in vivo. In conclusion, we presented a novel, more personalized and effective culture media for bovine IVP embryos. And although the ECS media formulation was based on the contents of female reproductive fluids, it is worth mentioning that adaptations must be specifically directed for in vitro conditions rather than reproduced exactly from in vivo state.

Evolutionary consequences of environmental effects on gamete performance.

Variation in pre- and post-release gamete environments can influence evolutionary processes by altering fertilization outcomes and offspring traits. It is now widely accepted that offspring inherit epigenetic information from both their mothers and fathers. Genetic and epigenetic alterations to eggs and sperm-acquired post-release may also persist post-fertilization with consequences for offspring developmental success and later-life fitness. In externally fertilizing species, gametes are directly exposed to anthropogenically induced environmental impacts including pollution, ocean acidification and climate change. When fertilization occurs within the female reproductive tract, although gametes are at least partially protected from external environmental variation, the selective environment is likely to vary among females. In both scenarios, gamete traits and selection on gametes can be influenced by environmental conditions such as temperature and pollution as well as intrinsic factors such as male and female reproductive fluids, which may be altered by changes in male and female health and physiology. Here, we highlight some of the pathways through which changes in gamete environments can affect fertilization dynamics, gamete interactions and ultimately offspring fitness. We hope that by drawing attention to this important yet often overlooked source of variation, we will inspire future research into the evolutionary implications of anthropogenic interference of gamete environments including the use of assisted reproductive technologies. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

The role of female reproductive fluid in sperm competition.

The role of non-gametic components of the ejaculate (seminal fluid) in fertility and sperm competitiveness is now well established. Surprisingly, however, we know far less about female reproductive fluid (FRF) in the context of sexual selection, and insights into male-FRF interactions in the context of sperm competition have only recently emerged. Despite this limited knowledge, evidence from taxonomically diverse species has revealed insights into the effects of FRF on sperm traits that have previously been implicated in studies of sperm competition. Specifically, through the differential effects of FRF on a range of sperm traits, including chemoattraction and alterations in sperm velocity, FRF has been shown to exert positive phenotypic effects on the sperm of males that are preferred as mating partners, or those from the most compatible or genetically diverse males. Despite these tantalizing insights into the putative sexually selected functions of FRF, we largely lack a mechanistic understanding of these processes. Taken together, the evidence presented here highlights the likely ubiquity of FRF-regulated biases in fertilization success across a diverse range of taxa, thus potentially elevating the importance of FRF to other non-gametic components that have so far been studied largely in males. This article is part of the theme issue 'Fifty years of sperm competition'.

Sperm Proteome after Interaction with Reproductive Fluids in Porcine: From the Ejaculation to the Fertilization Site.

Ejaculated sperm are exposed to different environments before encountering the oocyte. However, how the sperm proteome changes during this transit remains unsolved. This study aimed to identify proteomic changes in boar sperm after incubation with male (seminal plasma, SP) and/or female (uterine fluid, UF; and oviductal fluid, OF) reproductive fluids. The following experimental groups were analyzed: (1) SP: sperm + 20% SP; (2) UF: sperm + 20% UF; (3) OF: sperm + 20% OF; (4) SP + UF: sperm + 20% SP + 20% UF; and (5) SP+OF: sperm + 20% SP + 20% OF. The proteome analysis, performed by HPLC-MS/MS, allowed the identification of 265 proteins. A total of 69 proteins were detected in the UF, SP, and SP + UF groups, and 102 proteins in the OF, SP, and SP + OF groups. Our results showed a higher number of proteins when sperm were incubated with only one fluid than when they were co-incubated with two fluids. Additionally, the number of sperm-interacting proteins from the UF group was lower than the OF group. In conclusion, the interaction of sperm with reproductive fluids alters its proteome. The description of sperm-interacting proteins in porcine species after co-incubation with male and/or female reproductive fluids may be useful to understand sperm transport, selection, capacitation, or fertilization phenomena.

Reproductive Outcomes and Endocrine Profile in Artificially Inseminated versus Embryo Transferred Cows.

The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients' hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves' weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calves.