The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment.

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 µM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.

Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model.

Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI-median fluorescence intensity-and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

Does Size Matter? Testicular Volume and Its Predictive Ability of Sperm Production in Rams.

Over the years, testicular volume has been used to evaluate the reproductive capacity of rams and the effects of different factors related to reproductive performance. The aim of this study was to determine the most suitable tool and formula to calculate testicular volume under field conditions to guarantee a more accurate determination of sperm production. First, testicles from 25 rams (n = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS). The accurate testicular volume (ATV) was calculated through water displacement. In addition, the sexual status of donor rams was evaluated during a period of four years in a reproduction center, and the three most crucial groups in terms of genetic value and seminal collections were studied in the second part of this experiment: ER-NBS (Elite rams during the non-breeding season), ER-BS-S (Elite rams with a standard frequency of seminal collection), and ER-BS-O (Elite rams with a high frequency of seminal collection). The total testicular volume (TTV), testosterone (T), and total spermatozoa obtained from two consecutive ejaculates in the same day (SPERM) were measured, and the relationship between SPERM and TTV and T was analyzed to predict SPERM. Although all published formulas revealed statistically significant differences (p ≤ 0.05) from the ATV, our proposed formula (ItraULE) (Testicular volume = L × W × D × 0.61) did not show significant differences. In the second part of the study, in the ER as a model donor ram for its high genetic value and high demand from farmers, TTV and T showed strong positive correlations with SPERM (r = 0.587, p = 0.007 NBS; r = 0.684, p = 0.001 BS-S; r = 0.773, p < 0.0001 BS-O). Moreover, formulas were established to predict SPERM in these practical scenarios. In conclusion, the use of ultrasonography and a new formula adapted to rams could improve the prediction of SPERM considering crucial factors such as season and semen collection frequency.

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa.

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection.

Bos taurus and Cervus elaphus as Non-Seasonal/Seasonal Models for the Role of Melatonin Receptors in the Spermatozoon.

Melatonin is crucial in reproduction due its antioxidant, hormonal, and paracrine action. Melatonin membrane receptors (MT1/MT2) have been confirmed on spermatozoa from several species, but functionality studies are scarce. To clarify their role in ruminants as reproductive models, bull (Bos taurus, non-seasonal) and red deer (Cervus elaphus, highly seasonal) spermatozoa were analyzed after 4 h of incubation (38 °C, capacitating media) in 10 nM melatonin, MT1/MT2 agonists (phenylmelatonin and 8M-PDOT), and antagonists (luzindole and 4P-PDOT). Motility and functionality (flow cytometry: viability, intracellular calcium, capacitation status, reactive oxygen species (ROS) production, and acrosomal and mitochondrial status) were assessed. In bull, MT1 was related to sperm viability preservation, whereas MT2 could modulate cell functionality to prevent excess ROS produced by the mitochondria; this action could have a role in modulating sperm capacitation. Deer spermatozoa showed resistance to melatonin and receptor activation, possibly because the samples were of epididymal origin and collected at the breeding season's peak, with high circulating melatonin. However, receptors could be involved in mitochondrial protection. Therefore, melatonin receptors are functional in the spermatozoa from bull and deer, with different activities. These species offer models differing from traditional laboratory experimental animals on the role of melatonin in sperm biology.

Melatonin affects red deer spermatozoa motility and physiology in capacitating and non-capacitating conditions.

Melatonin affects sperm physiology, possibly through membrane receptors. Effects were tested at low concentrations (1 pM, 100 pM, 10 nM and 1 µM) in red deer epididymal spermatozoa as a model for high-seasonality species. Samples were incubated with melatonin as uncapacitated or capacitating conditions (heparin) and evaluated for motility and physiology (flow cytometry). Most effects occurred at low concentrations (nM-pM), mainly protecting from apoptosis and maintaining acrosomal integrity, suggesting a role for membrane receptors rather than a direct antioxidant effect. Intracellular calcium was not affected, differing from other studies and perhaps because of the epididymal origin. This study supports the relevance of melatonin on sperm physiology and could contribute to the application of reproductive technologies in wild ruminants.

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets.

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Effect of Season and Parity on Reproduction Performance of Iberian Sows Bred with Duroc Semen.

The Iberian pig is an autochthonous breed from the Iberian Peninsula highly valued for its meat. The sows are often bred as Iberian × Duroc crossings for increased efficiency. Since sow parity and season affect the reproductive performance, we evaluated two-year records from a commercial farrow-to-finish farm (live, stillborn, and mummified piglets after artificial insemination, AI). A total of 1293 Iberian sows were inseminated with semen from 57 boars (3024 AI). The effects of parity (gilts, 1, 2-4, 5-10, and >10 farrowings) and season were analyzed by linear mixed-effects models (LME). The data were fitted to cosinor models to investigate seasonal effects within parity groups. The effects of maximum daily temperature (MDT) and day length change (DLC) during spermatogenesis, pre-AI, and post-AI periods were analyzed with LME. The 2-4 group was the optimal one for parity. A seasonal effect was evident between spring-summer (lower fertility/prolificacy) and autumn-winter (higher). Cosinor showed that the seasonal drop in reproductive performance occurs earlier in Iberian sows than in other breeds, more evident in gilts. MDT negatively affected performance in all periods and DLC in spermatogenesis and pre-AI. These results are relevant for the improvement of Iberian sows' intensive farming.

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.

Nitrite and Nitrate Levels in Follicular Fluid From Human Oocyte Donors Are Related to Ovarian Response and Embryo Quality.

Nitric oxide, a key regulatory molecule in the follicular fluid, has been suggested as a possible biomarker to predict ovarian response in stimulated cycles and the potential of the retrieved oocytes for developing high-quality embryos. Nevertheless, a consensus on whether or not nitric oxide can help in this context has not been reached. We simultaneously measured the oxidation products of nitric oxide, nitrite, and nitrate, via high-performance liquid chromatography (HPLC)-UV in follicular fluid samples from 72 oocyte donors. We found no associations of follicular fluid nitrite, nitrate, total nitric oxide, or nitrate/nitrite ratio with total or metaphase II (MII) oocyte yield. However, nitrite and nitrate levels were related to the yield of MII oocytes when this outcome was expressed as a proportion of all oocytes retrieved. The adjusted MII proportion in the lowest and highest nitrite levels were 68% (58-77%) and 79% (70-85%), respectively (p, linear trend = 0.02), whereas the adjusted MII proportion in extreme tertiles of nitrate levels were 79% (70-85%) and 68% (57-77%) (p, linear trend = 0.03). In addition, nitrate levels showed a suggestive inverse correlation with embryos with maximum or high potential of implantation (p = 0.07). These results suggest that the follicular fluid concentrations of nitrite and nitrate may be a useful tool in predicting how healthy oocyte donors respond to superovulation and the implantation potential of the embryos produced from their oocytes.

Involvement of nitric oxide during in vitro oocyte maturation, sperm capacitation and in vitro fertilization in pig.

The importance of porcine species for meat production is undeniable. Due to the genetic, anatomical, and physiological similarities with humans, from a biomedical point of view, pig is considered an ideal animal model for the study and development of new therapies for human diseases. The in vitro production (IVP) of porcine embryos has become widespread as a result of these qualities and there is significant demand for these embryos for research purposes. However, the efficiency of porcine embryo IVP remains very low, which hinders its use as a model for research. The high degree of polyspermic fertilization is the main problem that affects in vitro fertilization (IVF) in porcine species. Furthermore, oocyte in vitro maturation (IVM) is another important step that could be related to polyspermic fertilization and low embryo production. The presence of nitric oxide synthase (NOS), the enzyme that produces nitric oxide (NO), has been detected in the oviduct, the ovary, the oocyte and the sperm cell of porcine species. Its functions include regulating oviductal activity, ovulation, acquisition of meiotic competence, oocyte activation, sperm capacitation, and gamete interaction. Therefore, in this review, we summarize the current knowledge on the role of NO/NOS system in each of the steps that lead to the production of porcine embryos in an in vitro environment, i.e. IVM, sperm capacitation, IVF, and embryo culture. We also discuss the possible ways in which the NO/NOS system could be used to enhance IVP of porcine embryos.

Epididymal and ejaculated sperm functionality is regulated differently by periovulatory oviductal fluid in pigs.

BACKGROUND: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. OBJECTIVES: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. MATERIAL AND METHODS: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyrosine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status. RESULTS: The pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte. CONCLUSION: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa.

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies.

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.

Effect of oviductal fluid on bull sperm functionality and fertility under non-capacitating and capacitating incubation conditions.

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation.

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.

Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants.

Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (p < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (p < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

BACKGROUND: Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. OBJECTIVES: This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. MATERIAL AND METHODS: The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). RESULTS: The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. CONCLUSION: OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.