Preliminary fertility results with frozen buffalo bull semen using tris extender

Various extenders have been proposed to freeze buffalo semen, but much remains to be done in terms of achieving optimum fertility. Fifty semen samples were frozen using tris extender. On an average, 54% and 48% post-freezing motility was observed after 15 minutes and one month storage, respectively. Seventy two buffaloes were inseminated with frozen semen stored at least one month. On the average, a 45.8% first service conception rate was obtained.     

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: Preliminary results

Glutamine has been used in the composition of semen extenders in several species, but never in the bull. The aim of our study is to demonstrate the cryoprotective role of glutamine for freezing bovine semen and to determine concentration of the latter to improve the motility and trajectory characteristics of spermatozoa. Three experiments were undertaken with 21 ejaculates from three different bulls. In the first experiment, glutamine was added to 40, 80, and 120 mM of basic medium (BM) which consisted of Tris + glycerol 6.4% (v/v). In the second experiment glutamine was added to the 8% low density lipoprotein (LDL) diluent at 40, 80, and 120 mM. In the third experiment, the best concentration of glutamine was determined; this was then added to the LDL extender at 10, 20, 30, and 40 mM. The semen was diluted then frozen in the different media. Motility parameters were assessed using an image analyser following thawing. Experiment 1 demonstrated that glutamine had a cryoprotective effect; at 40 mM it gave superior motility parameters to those obtained with the basic medium (p < 0.05). Experiment 2 demonstrated that the combination of LDL-glutamine 40 mM and 80 mM did not improve motility and even deteriorated it in comparison with the glutamine-free LDL extender. Experiment 3 demonstrated that the addition of 10 mM of glutamine to the LDL medium lead to a significant improvement (p < 0.05) in the motility of bull spermatozoa and could be used to improve bovine semen extenders.     

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender

Low-density lipoproteins (LDL) have been previously isolated and identified as the cryoprotective fraction of yolk. The effect of LDL on sperm motility after freezing-thawing has been reported, but no study has been made to assess the effect of LDL on bull semen fertility. The aim of this study was to evaluate the fertility of bull semen cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Optidyl, a commercial extender containing egg yolk. To evaluate the fertilizing ability of semen, we used in vitro fertilization test, whereas acrosome and plasma membrane integrity were also evaluated. The percentage of motile spermatozoa was two fold higher after freezing in LDL than in Optidyl 54.4% versus 30.2% (P < 0.05). The cleavage rate was significantly higher after fertilization with semen frozen in LDL than with Optidyl 63.0% versus 54.8% (P < 0.05). No significant difference was observed on the blastocyst rate after in vitro culture. Integrity of the acrosome and the plasma membrane were maintained in both extenders. In conclusion, LDL preserve bull semen quality and fertilizing ability, allowing also better semen motility, after the freeze-thaw process.     

LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake

Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.     

Improvement of the quality of boar cryopreservation semen by supplementing with low density lipoprotein in diluents

Low density lipoprotein was added at concentrations of 6%, 7%, 8%, 9% and 10% to the diluents used to freeze boar semen and its effect on the quality of cryopreservation was assessed The results indicated that 9% low density lipoprotein supplementation significantly improved the total motile sperm (p<0.05). The sperm straight-line velocity increased with the concentration of low density lipoprotein except at the concentration of 10%, at which concentration the sperm velocity declined (p<0.05). Supplementation at 8% and 9% low density lipoprotein significantly increased plasma membrane integrity (p<0.05). Compared with control, the low density lipoprotein supplementation significantly improved the percentage of acrosome integrity (p<0.05). With all parameters measured, the concentration of 9% low density lipoprotein showed a better effect on the quality of boar cryopreservation semen. The sperm DNA was more seriously damaged in the species of Yorkshire than in Duroc.     

Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL

Low density lipoprotein (LDL) particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. In the process of atherogenesis these particles are modified and they accumulate in the arterial wall. Although the composition and overall structure of the LDL particles is well known, the fundamental molecular interactions and their impact on the structure of LDL particles are not well understood. Here, the existing pieces of structural information on LDL particles are combined with computer models of the individual molecular components to give a detailed structural model and visualization of the particles. Strong evidence is presented in favor of interactions between LDL lipid constituents that lead to specific domain formation in the particles. A new three-layer model, which divides the LDL particle into outer surface, interfacial layer, and core, and which is capable of explaining some seemingly contradictory interpretations of molecular interactions in LDL particles, is also presented. A new molecular interaction model for the beta-sheet structure and phosphatidylcholine headgroups is introduced and an overall view of the tertiary structure of apolipoprotein B-100 in the LDL particles is presented. This structural information is also utilized to understand and explain the molecular characteristics and interactions of modified, atherogenic LDL particles.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.     

Association of apolipoprotein B gene variants with plasma apoB and low density lipoprotein (LDL) cholesterol levels

Summary The contribution of the variants of the apolipoprotein (apo) B locus to the total variance in plasma apoB and cholesterol levels was examined in four independent populations, two that were composed of normal controls (n = 77 and 85) and two with coronary heart disease (n = 115 and 159). A correlation between genotype at the apoB-XbaI locus and apoB levels was observed. The effects of the (+; presence of restriction site) and (-) alleles were to increase or decrease the apoB and cholesterol levels by approximately 3.5 mg/dl, respectively. None of the 274 individuals in the coronary heart disease (CHD) groups was found to be a carrier of the apoB allele Arg₃₅₀₀Gln, previously shown to be associated with an apoB protein defective in binding to the low density lipoprotein receptor (LDL-R). No DNA sequence variants were found in the region encoding amino acid residues 3129–3532 within the putative LDL-R binding domain among 35 individuals with apoB levels above the 94th percentile (141 mg/dl).     

Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison)

The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

The advantages of LDL (Low Density Lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

Human breast milk stimulates low density lipoprotein (LDL) binding to human skin fibroblasts in culture

Human breast milk incorporated at 1% concentration into the culture medium significantly (p less than 0.05) increased the binding of 125I-LDL to receptors of human skin fibroblasts in culture. Homogenized cows milk and infant formula (Similac) also possessed this stimulating property. The stimulating activity of milk persisted after dialysis and extraction with cold acetone. These preliminary studies suggest that milk might contain potent factor(s) influencing cholesterol metabolic process in early life.