| 1. |
- Abe, Y, et al.
(författare)
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Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts
- 2005
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Ingår i: Biology of Reproduction. - : Society for the Study of Reproduction. - 0006-3363 .- 1529-7268. ; 72:6, s. 1416-1420
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Tidskriftsartikel (refereegranskat)abstract
- To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P less than 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.
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| 2. |
- Al-Makhzoomi, A., et al.
(författare)
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Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden
- 2008
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 70:4, s. 682-691
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Tidskriftsartikel (refereegranskat)abstract
- Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n = 8) and Swedish Holstein (SLB, n = 4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n = 3) or pooling two consecutive ejaculates (n = 104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing greater than 10% of morphologically deviating sperm head shapes, a commonly used threshold for young At bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires. (c) 2008 Elsevier Inc. All rights reserved.
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| 3. |
- Alkmin, Diego V., et al.
(författare)
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Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
- 2014
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Ingår i: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 69:2, s. 203-210
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Tidskriftsartikel (refereegranskat)abstract
- Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17 degrees C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p less than0.01) than BE samples. Control samples showed higher (p less than 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p less than 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
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| 4. |
- Alminana, C, et al.
(författare)
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Adjustments in IVF system for individual boars: Value of additives and time of sperm-oocyte co-incubation
- 2005
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 64:8, s. 1783-1796
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Tidskriftsartikel (refereegranskat)abstract
- In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5943 COCs were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa froth 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2 mM), hyaluronic acid (HA; [0.5 mg/mL]) and adenosine (10 mu M), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5 mg/ml) and adenosine (0, 10, 20 and 40 mu M) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 thin or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6 h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 1215 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P less than 0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9 +/- 3.9% versus 62.7 +/- 3.9% and 1.5 +/- 3.2 versus 1.3 +/- 3.5 for 10 min or 6 h, respectively), but reduced monospermy (P less than 0.001, 57.9 +/- 2.5% versus 70.0 +/- 2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF. (c) 2005 Elsevier Inc. All rights reserved.
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| 5. |
- Alvarez-Rodriguez, Manuel, et al.
(författare)
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Chicken seminal fluid lacks CD9-and CD44-bearing extracellular vesicles
- 2020
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Ingår i: Reproduction in domestic animals. - : Wiley-VCH Verlagsgesellschaft. - 0936-6768 .- 1439-0531. ; 55:3, s. 293-300
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Tidskriftsartikel (refereegranskat)abstract
- The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.
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| 6. |
- Alvarez-Rodriguez, Manuel, et al.
(författare)
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Does the Act of Copulation per se, without Considering Seminal Deposition, Change the Expression of Genes in the Porcine Female Genital Tract?
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:15
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Tidskriftsartikel (refereegranskat)abstract
- Semen-through its specific sperm and seminal plasma (SP) constituents-induces changes of gene expression in the internal genital tract of pigs, particularly in the functional sperm reservoir at the utero-tubal junction (UTJ). Although seminal effects are similarly elicited by artificial insemination (AI), major changes in gene expression are registered after natural mating, a fact suggesting the act of copulation induces per se changes in genes that AI does not affect. The present study explored which pathways were solely influenced by copulation, affecting the differential expression of genes (DEGs) of the pre/peri-ovulatory genital tract (cervix, distal uterus, proximal uterus and UTJ) of estrus sows, 24 h after various procedures were performed to compare natural mating with AI of semen (control 1), sperm-free SP harvested from the sperm-peak fraction (control 2), sperm-free SP harvested from the whole ejaculate (control 3) or saline-extender BTS (control 4), using a microarray chip (GeneChip(R)porcine gene 1.0 st array). Genes related to neuroendocrine responses (ADRA1,ADRA2,GABRB2,CACNB2), smooth muscle contractility (WNT7A), angiogenesis and vascular remodeling (poFUT1,NTN4) were, among others, overrepresented with distal and proximal uterine segments exhibiting the highest number of DEGs. The findings provide novel evidence that relevant transcriptomic changes in the porcine female reproductive tract occur in direct response to the specific act of copulation, being semen-independent.
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| 7. |
- Alvarez-Rodriguez, Manuel, 1983-, et al.
(författare)
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Exogenous Individual Lecithin-Phospholipids (Phosphatidylcholine and Phosphatidylglycerol) Cannot Prevent the Oxidative Stress Imposed by Cryopreservation of Boar Sperm.
- 2017
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Ingår i: Journal of veterinary medicine and surgery. - : imedpub. - 2574-2868. ; 1:1
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Despite the use of high proportions of the chemically undefined lipoprotein/phospholipid-rich egg-yolk in extenders, boar sperm are highly sensitive to cooling, which induces ROS generation and disrupts the plasma membrane.Here, we studied whether replacement of hen egg-yolk by commercially defined lecithin phospholipids, derived from egg (LPGE: phosphatidyl glycerol, LPCE: phosphatidyl choline) or soybean (LPCS: phosphatidyl choline), could individually ameliorate such oxidative effects during cryopreservation of ejaculated (sperm rich fraction, SRF) or of cauda-epididymal sperm, retrieved post-mortem from the same males.Methods: A conventional extender (lactose buffer, with 20% egg-yolk, 0.5% OEP and 3% glycerol) was used as control. Cryodamage was assessed as loss of sperm motility, membrane and acrosome intactness, early membrane destabilization changes, mitochondrial potential, superoxide and ROS production, to finally determine lipid peroxidation (LPO) using specific probes.Results and conclusion: In general, the exogenous phospholipids assayed were unable of maintaining neither sperm motility nor viability post-thaw compared to controls, owing to increased ROS production and lipid peroxidation. In our study, mitochondrial superoxide production resulted in very high levels for all groups, whereas both ROS production and lipid peroxidation were reduced in the control group, containing emulsified hen egg yolk. Further studies using various dosage and combination of LPCS should be followed for their eventual protective effect.Keywords: Cryodamage; Sperm; Boar; Mitochondrial activation; Mitochondrial superoxide; ROS production; Lipid peroxidation
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| 8. |
- Alvarez-Rodriguez, Manuel, et al.
(författare)
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Exosomes in specific fractions of the boar ejaculate contain CD44: A marker for epididymosomes?
- 2019
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Ingår i: Theriogenology. - : ELSEVIER SCIENCE INC. - 0093-691X .- 1879-3231. ; 140, s. 143-152
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Tidskriftsartikel (refereegranskat)abstract
- Seminal plasma (SP) is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles (EVs). The SP interacts with spermatozoa and the inner cell lining of the female genital tract, adsorbing proteins and exosomes that modulate sperm functions and female immune responsiveness. In the present study, boar sperm-free SP was studied using flow cytometry (FC) after membrane tetraspanins (CD9, CD63 and CD81) and membrane receptor CD44 marking of non-enriched (whole SP) or gradient fractions enriched through two-step discontinuous KBr-density-gradient ultracentrifugation, in whole ejaculate or in selected ejaculate fractions. The results, evaluated by transmission electron microscopy, confirmed the presence of exosomes in all fractions of the pig SP. Noteworthy, these pig SP-exosomes were CD44-bearing when analysed by FC, with bands detected by western blotting (WB) at the expected 85 kD size. The two-step discontinuous KBr-density-gradient ultracentrifugation enriched the population of exosomes in two specific gradient fractions, indicating exosomes (either prostasomes or epididymosomes) could be separated from low-density lipoprotein (LDL) but they co-sediment with the high-density lipoprotein (HDL)-bearing fraction. The findings pave for the selective isolation of exosomes in functional studies of their function when interacting with spermatozoa, the oocyte and/or the female genitalia, including hyaluronan-CD44 interplay. (C) 2019 Elsevier Inc. All rights reserved.
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| 9. |
- Alvarez-Rodriguez, Manuel, 1983-, et al.
(författare)
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Expression of Immune Regulatory Genes in the Porcine Internal Genital Tract Is Differentially Triggered by Spermatozoa and Seminal Plasma
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 20:3
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Tidskriftsartikel (refereegranskat)abstract
- Mating or cervical deposition of spermatozoa or seminal plasma (SP) modifies the expression of genes affecting local immune defense processes at the oviductal sperm reservoir in animals with internal fertilization, frequently by down-regulation. Such responses may occur alongside sperm transport to or even beyond the reservoir. Here, immune-related gene expression was explored with cDNA microarrays on porcine cervix-to-infundibulum tissues, pre-/peri-ovulation. Samples were collected 24 h post-mating or cervical deposition of sperm-peak spermatozoa or SP (from the sperm-peak fraction or the whole ejaculate). All treatments of this interventional study affected gene expression. The concerted action of spermatozoa and SP down-regulated chemokine and cytokine (P00031), interferon-gamma signaling (P00035), and JAK/STAT (P00038) pathways in segments up to the sperm reservoir (utero-tubal junction (UTJ)/isthmus). Spermatozoa in the vanguard sperm-peak fraction (P1-AI), uniquely displayed an up-regulatory effect on these pathways in the ampulla and infundibulum. Sperm-free SP, on the other hand, did not lead to major effects on gene expression, despite the clinical notion that SP mitigates reactivity by the female immune system after mating or artificial insemination.
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| 10. |
- Alvarez-Rodriguez, Manuel, et al.
(författare)
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Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing Al boar spermatozoa
- 2018
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Ingår i: Journal of reproduction and development. - : SOCIETY REPRODUCTION & DEVELOPMENT-SRD. - 0916-8818 .- 1348-4400. ; 64:4, s. 351-360
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan (hyaluronic acid, HA) apparently improves sperm survival in vitro and in vivo (oviduct), maintaining sperm motility and inducing capacitation, but not acrosome exocytosis, either by direct action as a macromolecule or via CD44 membrane receptors. This study explored ejaculated, liquid-extended pig spermatozoa to ascertain (i) the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor, using a specific monoclonal commercial antibody; (ii) whether the CD44 receptor changed location when exposed to bicarbonate, a capacitating trigger, in vitro; and (iii) whether the addition of HA, of molecular size comparable to that produced in the oviduct sperm reservoir (0.0625 to 2.0 mg/ml; 0 HA: control), to semen extenders would improve sperm liquid storage in vitro or cryosurvival post freezing. Variables tested were sperm velocity and progressive motility (Qualisperm (TM)), sperm viability and acrosome status, membrane integrity and early destabilization, mitochondrial activation, and superoxide production (flow cytometry). The CD44 receptor presence in ejaculated, liquid-stored AI boar spermatozoa, as confirmed by a porcine-specific monoclonal antibody, maintained its membrane location under in vitro capacitation-inducing conditions. HA exposure to 24-, 48-, or 72-h liquid-stored (17-20 degrees C) spermatozoa lowered sperm velocity in membrane-intact spermatozoa, but increased mitochondrial superoxide production. Finally, HA addition during cooling did not improve cryosurvival but did increase mitochondrial activation and membrane destabilization in surviving cells. These results confirm the existence of a CD44 receptor in pig spermatozoa, but the usefulness of adding HA for long-term storage or cryopreservation of liquid-stored, extended boar semen remains in question, thereby warranting further non-empirical analyses of HA-sperm membrane interactions.
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