Reduction of cortical pulling at mitotic entry facilitates aster centration.

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

Early Pregnancy Markers in the Serum of Ewes Identified via Proteomic and Metabolomic Analyses.

The diagnosis of ewes' pregnancy status at an early stage is an efficient way to enhance the reproductive output of sheep and allow producers to optimize production and management. The techniques of proteomics and metabolomics have been widely used to detect regulatory factors in various physiological processes of animals. The aim of this study is to explore the differential metabolites and proteins in the serum of pregnant and non-pregnant ewes by proteomics and metabolomics. The serum of ewes at 21, 28 and 33 days after artificial insemination (AI) were collected. The pregnancy stratus of the ewes was finally determined through ultrasound examination and then the ewes were grouped as Pregnant (n = 21) or N on-pregnant (n = 9). First, the serum samples from pregnant or non-pregnant ewes at 21 days after AI were selected for metabolomic analysis. It was found that the level of nine metabolites were upregulated and 20 metabolites were downregulated in the pregnant animals (p < 0.05). None of these differential metabolomes are suitable as markers of pregnancy due to their small foldchange. Next, the proteomes of serum from pregnant or non-pregnant ewes were evaluated. At 21 days after AI, the presence of 321 proteins were detected, and we found that the level of three proteins were upregulated and 11 proteins were downregulated in the serum of pregnant ewes (p < 0.05). The levels of serum amyloid A (SAA), afamin (AFM), serpin family A member 6 (SERPINA6) and immunoglobulin-like domain-containing protein between pregnant and non-pregnant ewes at 21-, 28- and 33-days post-AI were also analyzed via enzyme-linked immunosorbent assay (ELISA). The levels of SAA and AFM were significantly higher in pregnant ewes than in non-pregnant ewes, and could be used as markers for early pregnancy detection. Overall, our results show that SAA and AFM are potential biomarkers to determine the early pregnancy status of ewes.

Metabolomic signature of spermatozoa established during holding time is responsible for differences in boar sperm freezability†.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.

Vitamin E promotes ovine Sertoli cell proliferation by regulation of genes associated with cell division and the cell cycle.

The effect of Vitamin E on the proliferation of ovine Sertoli cells was investigated. Sertoli cells were isolated and treated with various amounts of Vitamin E (0 µM, 400 µM, 800 µM, 1000 µM, 1200 µM, 1400 µM and 1600 µM) for 24 h. We found that at the concentration of 1200 µM, Vitamin E promoted Sertoli cell proliferation very effectively. It also increased the proportion of cells in the G1 phase while reduced that in the S and G2/M phases, suggesting that its effect on Sertoli cell proliferation is achieved by enhancing progression through the cell cycle. In addition, Vitamin E significantly up-regulated the transcript level of the PDPN, BMP6, AMPKα, GSK3ß, Myc, and CDK6 genes and down-regulated that of PPARγ, Cyclin B1 and CDK4 as determined by qRT-PCR. Western blot analysis revealed that the expression of BMP6 and PDPN was also upregulated at the protein level, in accordance with the results of the qRT-PCR. Taken together, Vitamin E promoted Sertoli cell proliferation by affecting the expression of genes that regulate cell division and the cell cycle; this indicates that it can have a positive effect on sheep reproductive performance.

Biodynamic digital holographic speckle microscopy for oocyte and embryo metabolic evaluation.

Assisted reproductive technologies seek to improve the success rate of pregnancies. Morphology scoring is a common approach to evaluate oocyte and embryo viability prior to embryo transfer in utero, but the efficacy of the method is low. We apply biodynamic imaging, based on dynamic light scattering and low-coherence digital holography, to assess the metabolic activity of oocytes and embryos. A biodynamic microscope, developed to image small and translucent biological specimens, is inserted into the bay of a commercial inverted microscope that can switch between conventional microscopy channels and biodynamic microscopy. We find intracellular Doppler spectral features that act as noninvasive proxies for embryo metabolic activity that may relate to embryo viability.

Back to the future: optimised microwell culture of individual human preimplantation stage embryos.

Although in vitro culture of human embryos is a crucial step in assisted reproduction, the lack of focused research hampers worldwide standardisation and consistent outcomes. Only 1.2% of research papers published in five leading journals in human reproduction in 2019 focused on in vitro culture conditions, creating the impression that the optimisation process has approached its limits. On the other hand, in vitro culture of mammalian embryos is based on old principles, while there is no consensus on basic issues as density, time, medium change, gas atmosphere and small technical details including the way of drop preparation. This opinion paper aims to highlight and analyse the slow advancement in this field and stimulate research for simple and affordable solutions to meet the current requirements. A possible way for advancement is discussed in detail. Selection of embryos with the highest developmental competence requires individual culture and modification of the widely used "drop under oil" approach. Current use of three-dimensional surfaces instead of large flat bottoms is restricted to time-lapse systems, but these wells are designed for optical clarity, not for the needs of embryos. The size and shape of the original microwells (Well of the Well; WOW) offer a practical and straightforward solution to combine the benefits of communal and individual incubation and improve the overall quality of cultured embryos.

Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1.

The Ca2+ entry mechanism that sustains the Ca2+ oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca2+ stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes' cytosolic free Ca2+ concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca2+ release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca2+ entry (SOCE) induced by Ca2+ add-back after a CPA treatment; it also disrupted the Ca2+ oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca2+ oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca2+ oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca2+ oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca2+ transients at fertilization.

Egg Activation at Fertilization.

Fertilization is the union of gametes to initiate development of a new individual. The female gamete is formed during oogenesis. The process begins when, in the early embryo, primordial germ cells arise and subsequently colonize the genital ridges. They differentiate into oogonia, start meiosis, and become primary oocytes. The cell cycle of the primary oocytes then becomes arrested in mid-meiosis for an extended period of time. Prior to ovulation the oocytes undergo a growth phase and their sizes increase significantly. A hormonal cue then triggers oocyte maturation that involves the resumption of meiosis, the completion of the first meiotic division, and, as a result, the reduction in the diploid chromosome number. The cell cycle then stops again; in vertebrates this arrest occurs at the metaphase stage of the second meiotic division. Meiosis resumes at fertilization, when the sperm activates the egg, i.e., it causes a series of changes that are required for the initiation of embryo development. This is achieved by triggering an elevation in the egg's intracellular free calcium concentration. In response, the fertilized egg completes meiosis and enters the first embryonic cell cycle.

Fertility: Store-Operated Ca2+ Entry in Germ Cells: Role in Egg Activation.

At the time of fertilization, the sperm activates the egg and induces embryonic development by triggering an elevation in the egg's intracellular free Ca2+ concentration. In mammals the initial Ca2+ rise is followed by a series of repetitive Ca2+ transients (known as oscillations) that last for several hours. Although the source of Ca2+ during the signaling process is primarily the egg's smooth endoplasmic reticulum, the oscillations stop in the absence of extracellular Ca2+ indicating that a Ca2+ influx across the plasma membrane is essential to sustain them. Depletion of the intracellular stores using specific inhibitors generates a Ca2+ entry across the plasma membrane of eggs of various species, and a continuous influx of Ca2+ has been linked to the sperm-induced Ca2+ oscillations in the mouse; these data indicate that store-operated Ca2+ entry (SOCE) operates in eggs and may be the mechanism that maintains the long-lasting Ca2+ signal at fertilization. Recent findings suggest that the signaling proteins STIM1 and Orai1 are present in eggs; they are responsible for mediating SOCE, and their functions are essential for proper Ca2+ signaling at fertilization to support normal embryo development.

The dynamics of MAPK inactivation at fertilization in mouse eggs.

Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos-luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.

Signal transduction in mammalian oocytes during fertilization.

Mammalian embryo development begins when the fertilizing sperm triggers a series of elevations in the oocyte's intracellular free Ca(2+) concentration. The elevations are the result of repeated release and re-uptake of Ca(2+) stored in the smooth endoplasmic reticulum. Ca(2+) release is primarily mediated by the phosphoinositide signaling system of the oocyte. The system is stimulated when the sperm causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 then binds its receptor on the surface of the endoplasmic reticulum that induces Ca(2+) release. The manner in which the sperm generates IP3, the Ca(2+) mobilizing second messenger, has been the subject of extensive research for a long time. The sperm factor hypothesis has eventually gained general acceptance, according to which it is a molecule from the sperm that diffuses into the ooplasm and stimulates the phosphoinositide cascade. Much evidence now indicates that the sperm-derived factor is phospholipase C-zeta (PLCζ) that cleaves PIP2 and generates IP3, eventually leading to oocyte activation. A recent addition to the candidate sperm factor list is the post-acrosomal sheath WW domain-binding protein (PAWP), whose role at fertilization is currently under debate. Ca(2+) influx across the plasma membrane is also important as, in the absence of extracellular Ca(2+), the oscillations run down prematurely. In pig oocytes, the influx that sustains the oscillations seems to be regulated by the filling status of the stores, whereas in the mouse other mechanisms might be involved. This work summarizes the current understanding of Ca(2+) signaling in mammalian oocytes.

The effects of permeating cryoprotectants on intracellular free-calcium concentrations and developmental potential of in vitro-matured feline oocytes.

Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca(2+)]i), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca(2+)]i, but changes in [Ca(2+)]i, resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (<5% for all treatments) were not affected (P>0.05) by extracellular Ca(2+). Exposure to EG (>44.1%) and DMSO (19.7%) increased (P<0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (≤2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P>0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage.

Store-Operated Ca2+ Entry Sustains the Fertilization Ca2+ Signal in Pig Eggs.

The role of store-operated Ca(2+) entry (SOCE) in the maintenance of sperm-induced Ca(2+) oscillations was investigated in porcine eggs. We found that 10 µM gadolinium (Gd(3+)), which is known to inhibit SOCE, blocked Ca(2+) entry that was triggered by thapsigargin-induced store depletion and also caused an abrupt cessation of the fertilization Ca(2+) signal. In a similar manner 3,5-bis(trifluoromethyl)pyrazole 2 (20 µM), and tetrapandin-2 (10 µM), potent SOCE inhibitors, also blocked thapsigargin-stimulated Ca(2+) entry and disrupted the Ca(2+) oscillations after sperm-egg fusion. The downregulation of Stim1 or Orai1 in the eggs did not alter the Ca(2+) content of the intracellular stores, whereas co-overexpression of these proteins led to the generation of irregular Ca(2+) transients after fertilization that stopped prematurely. We also found that thapsigargin completely emptied the endoplasmic reticulum, and that the series of Ca(2+) transients stopped abruptly after the addition of thapsigargin to the fertilized eggs, indicating that the proper reloading of the intracellular stores is a prerequisite for the maintenance of the Ca(2+) oscillations. These data strengthen our previous findings that in porcine eggs SOCE is a major signaling cascade that is responsible for sustaining the repetitive Ca(2+) signal at fertilization.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

STIM1 is required for Ca2+ signaling during mammalian fertilization.

During fertilization in mammals, a series of oscillations in the oocyte's intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocyte's intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes.

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.

The dynamics of PKC-induced phosphorylation triggered by Ca2+ oscillations in mouse eggs.

Fertilization of mammalian eggs is characterized by a series of Ca(2+) oscillations triggered by a phospholipase C activity. These Ca(2+) increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC-induced phosphorylation in mouse eggs, alongside Ca(2+) oscillations, using fluorescent C-kinase activity reporter (CKAR) probes. Ca(2+) oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr(2+) all caused repetitive increases in PKC-induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca(2+) transients. High frequency oscillations caused by PLCζ led to an integration of PKC-induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca(2+) oscillations using an isoform-specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ-specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC-induced phosphorylation outlasts each Ca(2+) increase in mouse eggs but that signal integration only occurs at a non-physiological, high Ca(2+) oscillation frequency. The results also suggest that Ca(2+) -induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization.

Calcium influx in mammalian eggs.

Calcium (Ca(2)(+)) signals are involved in the regulation of oocyte maturation and play a critical role during fertilization. In the egg, Ca(2)(+) is stored in the lumen of the endoplasmic reticulum and a signal is generated when the stored Ca(2)(+) is released through specialized channels in the membrane of the endoplasmic reticulum to elevate the free Ca(2)(+) concentration in the cytoplasm. Extracellular Ca(2)(+) is also important, indicated by the fact that the mobilization of luminal Ca(2)(+) is typically followed by Ca(2)(+) entry across the plasma membrane. The transmembrane Ca(2)(+) flux replenishes the endoplasmic reticulum, and thus, it is essential to sustain prolonged Ca(2)(+) signals. It also seems to be responsible for the stimulation of important signaling cascades required for complete egg activation. Characterization of the pathway that mediates Ca(2)(+) entry implies that its major components include STIM1, a protein that senses the filling status of the stores, and ORAI1, a channel protein located in the plasma membrane. Defining the mechanism and functions of Ca(2)(+) entry will not only lead to a better understanding of egg physiology but may also help improving the efficiency of a number of assisted reproductive technologies.

Production of Cloned Pigs by Handmade Cloning.

Somatic cell nuclear transfer (SCNT) in pigs is a promising technology in biomedical research by association with transgenesis for xenotransplantation and disease modeling technologies. Handmade cloning (HMC) is a simplified SCNT method that does not require micromanipulators and facilitates the generation of cloned embryos in large quantities. As a result of HMC fine-tuning for porcine-specific requirements of both oocytes and embryos, HMC has become uniquely efficient (>40% blastocyst rate, 80-90% pregnancy rates, 6-7 healthy offspring per farrowing, and with negligible losses and malformations). Therefore, this chapter describes our HMC protocol to obtain cloned pigs.

Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa.

This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 °C to the freezing lab. On arrival, samples were split into the treatments with the following additions before cryopreservation; 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Semen was evaluated for motility kinetics at 30, 120, and 240 min after thawing. Flow cytometry assessments were performed at 60 min after thawing. At all-time points evaluated, total and progressive motility were greater (P ≤ 0.05) in semen cryopreserved with GSH than in Control. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity; however, the proportion of capacitated spermatozoa were reduced (by -21.17%) in semen treated with BHT + GSH compared to Control (P ≤ 0.05). In contrast, there was a higher (P ≤ 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. For IVF, semen cryopreserved with both antioxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (-54.11%) compared to Control. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P ≤ 0.05) for BHT compared to Control. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment.