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PubMed2006

Ways to improve the biosecurity of bovine semen.

Semen production and trade is a worldwide industry. A framework, based on international standards is awaiting international and national regulation. In the perspective of biosecurity of the final product, critical notes can be made according to the semen production regulation and product safety. Process description brings the obligatory health standards for the production bulls, collection and processing of semen, identification, registration, worldwide distribution and insemination into discussion. Test frequency, test quality and demands, way of sampling and test consistency can influence product safety. New scientific knowledge can influence the value of the regulation. Whether a country is free of notifiable disease should influence decisions regarding necessary tests for the production bulls. The biosecurity of the semen production process is influenced by several factors. The effectiveness of the antibiotics used is questionable. The extenders for cryopreservation added to the semen can affect product safety. The way materials and storage containers have to be disinfected must be clear. In modern industry, tracking and tracing is an important issue. Worldwide differences in ways of identification of straws do not benefit a proper identification and registration throughout the process. Regulation could help improve the transparency of production and trade. Before anything concerning biohazard is implemented in regulation, each rule should be thoroughly based on scientific research where possible and furthermore it must be possible to enforce the regulation. The effort it takes to enforce the regulation should be in balance with the benefit it provides. An approach to alter regulation quickly is advisable. To produce a safe product that is accepted for international trade is of vital interest for the survival of artificial insemination (AI) in cattle.

Animal Identification Systems↗
PubMedDate not supplied

In vitro propagation of two Perkinsus spp. parasites from Japanese Manila clams Venerupis philippinarum and description of Perkinsus honshuensis n. sp.

Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, and Spain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April 2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate- and high-intensity infections. Perkinsus sp. cells in tissue samples were enlarged in alternative Ray's fluid thioglycollate medium, before propagation in DME:Ham's F-12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, which gave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolates comprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morphotype, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes, were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of all described Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents a new species, Perkinsus honshuensis n. sp.

Animals↗
PubMed2003

Detection of HLA-G on human extravillous cytotrophoblast and skeletal muscle with a new monoclonal antibody MEM-G/1.

Using immunohistochemistry with the newly available monoclonal antibody MEM-G/1 the reaction patterns on frozen and formaldehyde-fixed paraffin-embedded sections on human placentas, lymph nodes, skeletal muscles, and kidney and liver allografts were compared. HLA-G (a nonclassical major histocompatibility complex class I molecule that is assumed to influence the immune response during pregnancy and some pathological conditions) was found within human extravillous cytotrophoblast but not within villous cytotrophoblast and placental mesenchymal tissue. No HLA-G expression on human lymph nodes, tonsils, and kidney and liver allografts was demonstrated. However, HLA-G expression was observed in all samples of skeletal muscle. The binding capacity of monoclonal antibody MEM-G/1 provides new possibilities to study physiological and pathophysiological roles of HLA-G in humans.

Animals↗
PubMed1997

High fertilization rate obtained after intracytoplasmic sperm injection with 100% nonmotile spermatozoa selected by using a simple modified hypo-osmotic swelling test.

OBJECTIVE: To report a high fertilization rate after intracytoplasmic sperm injection (ICSI) in patients with 100% nonmotile spermatozoa selected by a simple modified hypo-osmotic swelling test. DESIGN: Clinical study. SETTING: Hospital-based IVF center. PATIENT(S): Three couples with infertility due to asthenospermia. INTERVENTION(S): The hypo-osmotic swelling test with 150-mOsm NaCl solution was used to select viable spermatozoa before ICSI. Three patients provided semen samples and one of these three also had a testicular biopsy. MAIN OUTCOME MEASURE(S): Selection of viable spermatozoa using the hypo-osmotic swelling test with 150-mOsm NaCl solution for ICSI. RESULT(S): No motile spermatozoa were found in three ejaculated semen samples and one testicular biopsy. Fifty-seven metaphase-II oocytes were injected with hypo-osmotic swelling test-positive ejaculated or testicular spermatozoa. Fifty-five (96.5%) of these oocytes were intact after injection. Forty-two (76.4%) of 55 oocytes showed two pronuclei, and 40 of the 42 fertilized oocytes cleaved. One patient had all embryos cryopreserved because of the risk of hyperstimulation; two other patients had embryos transferred. One ongoing pregnancy resulted. CONCLUSION: This hypo-osmotic swelling test with 150-mOsm NaCl solution is a simple and efficient method for selection of viable spermatozoa. A high fertilization rate can be obtained using ICSI with viable spermatozoa selected by using this hypo-osmotic swelling test.

Cryopreservation↗
PubMedDate not supplied

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms.

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.

Colony Count, Microbial↗
PubMed2002

Effects of frozen storage on the recovery of clenbuterol from urine samples for official control.

The effects of storage under frozen conditions on clenbuterol recovery from spiked calf urine samples were studied. Urine samples contaminated with 10 micro g l(-1) clenbuterol and frozen at -15 degrees C were analysed every 15 days over 6 months. A single frozen contaminated urine sample was also thawed every 15 days over 3 months for the analysis of 10-ml aliquots, after which the remaining portion was frozen again (-15 degrees C). The presence of clenbuterol was determined by GC-MS. A gradual decline in clenbuterol recovery was observed from the first to the 180th day. It was only possible to recover 1.74 +/- 0.06 micro g l(-1) (17%) clenbuterol on the 180th day in samples stored at -15 degrees C. Likewise, clenbuterol totally disappeared from spiked urine samples that had been successively frozen and thawed over 3 months. The results were confirmed in a study performed on 2704 calf urine samples collected on farms and analysed by HPTLC and GC-MS. Of 73 positive samples, 61 had been frozen for <10 days.

Adrenergic beta-Agonists↗
PubMed2004

PCR-detected hepatitis C virus RNA associated with human zona-intact oocytes collected from infected women for ART.

BACKGROUND: The aim of this study was to investigate the susceptibility of human oocytes from hepatitis C virus (HCV) RNA-positive women to HCV contamination during assisted reproductive technology (ART). METHODS: A reverse transcriptase-PCR assay was used to test for the presence of HCV RNA associated with 24 unfertilized oocytes 48 h after follicular fluid aspiration in 10 IVF attempts (seven conventional IVF and three ICSI). Negative and positive controls (10 unfertilized oocytes from HCV-negative women and 20 unfertilized oocytes artificially contaminated with HCV RNA-positive plasma; HCV RNA was also quantified in plasma and follicular fluid) were included. RESULTS: HCV RNA was associated with 17/24 (70.8%) oocytes (6/7 after ICSI and 11/17 after conventional IVF) and was found in 19/20 (95%) follicular fluid samples. A weak correlation was found between plasma and follicular fluid HCV RNA loads (r = 0.73, P < 0.001). CONCLUSIONS: HCV associated with unfertilized oocytes surrounded by their intact zona pellucida from anti-HCV antibody-positive and viraemic women undergoing ART raises questions concerning the safe management of medically assisted procreation for these women and good practice of oocyte/embryo cryopreservation and donation.

Adult↗
PubMed2000

Retinol analysis in dried blood spots by HPLC.

There are many advantages to measuring vitamin A in dried blood spots (DBS) from a finger prick as compared to plasma collected by venipuncture. The advantages include easier collection, transport and storage; accessibility to younger and more remote populations; and decreased risk of disease transmission. We describe a method for the extraction of retinol from DBS for analysis by HPLC and initial comparison to plasma retinol. The effects of various buffers, detergents, antioxidants and chelators were evaluated to establish the most effective approach to elute the retinol: retinol binding protein (holo-RBP) complex from the blood collection cards. The process involves ultrasonic agitation to elute holo-RBP into a phosphate buffer containing an antioxidant and metal chelator. The holo-RBP complex was denatured by the addition of ethanol containing additional antioxidants permitting the extraction of free retinol into hexane. Following solvent evaporation, the extract was dissolved in methanol for HPLC analysis. The initial measured retinol levels in freshly collected DBS declined for 6-10 d whether stored at 25, 4 or -20 degrees C, but remained consistent thereafter (homeostatic). By incorporating a "recovery/volume adjustment" factor, measured retinol values in homeostatic DBS were adjusted to the equivalent of plasma retinol. For 17 normal adults, the correlation coefficient was 0.90 between plasma retinol and adjusted DBS retinol in samples that had been stored at -70 degrees C for < 9 mo. The use of this new sample matrix for vitamin A assessment will allow access to previously unavailable populations.

Adult↗
PubMed1998

Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Long-term cryoconservation and stability of vitamin C in serum samples of the European prospective investigation into cancer and nutrition.

Plasma vitamin C level may be associated with risk of some chronic diseases. The rapid degradability of vitamin C in biological samples necessitates its stabilization with metaphosphoric acid or similar agents. However, in most cohort studies, prospectively collected biological samples are not treated with stabilizing agents before long-term frozen storage and it is not known whether vitamin C can be properly measured in such samples. The objective of this study was to determine the degree of vitamin C degradation in plasma samples stored without stabilization for 7 to 11 years at -196 degrees C. Spearman's correlation coefficients indicate a moderate correlation between baseline and final plasma vitamin C levels in both men (r = 0.57, P < 0.0001) and women (r = 0.52, P < 0.0001). Samples were also categorized based on low or high baseline levels of plasma vitamin C, with the latter category showing the highest rate of loss per year of frozen storage in men (1.96 micromol/L, P value for difference <0.0001; percent loss 24.6%) and women (2.35 micromol/L, P value for difference <0.0001; percent loss 24.2%), as determined by multiple regression analysis adjusted for smoking status, age, and body mass index. In men, both baseline and final plasma vitamin C values were lower in smokers than never smokers, but for both men and women the rate of vitamin C loss during storage was not significantly different between smokers and never smokers. The results of this study show that vitamin C can be measured with reasonable reliability in plasma samples frozen for long periods of time without addition of any stabilizing agents.

Antioxidants↗
PubMed2005

Optimization of the conditions for preservation of induced sputum: influence of freezing on cellular analysis.

BACKGROUND: Induced sputum (IS) analysis is a noninvasive, valid, and reproducible method for evaluating airway inflammation. It has been suggested that freezing of IS samples in order to delay analysis is feasible. However, the optimal conditions for preservation of IS samples have not been determined. OBJECTIVES: To determine optimal freezing conditions of IS samples, ensuring adequate specimen quality for assessment of cell viability, total cell count, and differential cell count. SUBJECTS AND METHODS: Twenty-one subjects were enrolled: 6 healthy control subjects, 5 patients with allergic rhinitis, 5 patients with mild asthma, and 5 patients with severe asthma. Each came to the laboratory once for IS sampling. Cell plugs were homogenized with dithiothreitol and separated into 12 aliquots. Viability and total and differential cell counts were determined for each aliquot. Bovine serum albumin (BSA) with dimethylsulfoxide (DMSO) was added to half of the aliquots, and fetal bovine serum (FBS) with DMSO was added to the other half. One half of the aliquots containing BSA or FBS were frozen at -20 degrees C, and the other half were frozen at - 80 degrees C. After 3, 7, or 10 days, samples were thawed and total cell counts, viability, and differential cell counts were assessed. RESULTS: Slide quality and total cell counts did not vary significantly according to freezing duration, temperature, or medium when compared to nonfrozen control samples. With FBS at -80 degrees C, cell viability did not vary significantly between control samples and freezing for 3, 7, and 10 days (59% vs 54%, 59% vs 54%, and 58% vs 54%, respectively; p > 0.05), whereas every other condition showed a significant decrease. Freezing did not affect the eosinophil percentage significantly. CONCLUSION: Freezing of IS samples in FBS with DMSO at - 80 degrees C allows adequate preservation of IS specimens. Samples can be kept for at least 10 days in those conditions without significantly altering total cell counts, viability, and eosinophil percentage.

Adolescent↗
PubMedDate not supplied

[Monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for Listeria monocytogenes control].

The monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for the Listeria monocytogenes control in the cooked and frozen meat section of a thermo-processing meat plant was evaluated. Seventy "non-product-contact" surface samples and fourteen finished product samples were examined. Thirty eight positive sites for the presence of Listeria sp. were obtained. Twenty-two isolates were identified as L. monocytogenes, two as L. seeligeri and fourteen as L. innocua. Non isolates were obtained from finished product samples. The detection of L. monocytogenes in cooked and frozen meat section environment showed the need for the HACCP plan to eliminate or prevent product contamination in the post-thermal step.

Animals↗
PubMed2006

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality.

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.

Acrosome↗
PubMed2005

Comparison of cryopreservation and standard needle biopsy for gene expression profiling of human breast cancer specimens.

Given the growing importance of molecular profiling of breast cancer, we initiated a small study to test whether human breast biopsies obtained via cryopreservation large core needle biopsy (C-LCNB) provided similar gene expression profiles compared with 'optimally handled' standard large core needle biopsy (S-LCNB) specimens. Five matched pairs of C-LCNB versus S-LCNB were obtained at the same visit, and subjected to gene array expression analysis using the Affymetrix system with U133A chips. No significant changes in gene expression were identified comparing the C-LCNB versus the matched S-LCNB from individual patients. This was corroborated by a paired t-test analysis, which supported the hypothesis that the S/C biopsies measured equivalent samples. A small number of genes (17) showed decreased expression when second biopsies were compared with first biopsies, suggesting a slight patient response to the first biopsy. A scatter plot analysis comparing first biopsy versus second biopsy values disclosed a slope of 0.859, further indicating that the first biopsy affects the second biopsy measurement. It therefore appears that conventional biopsies, when handled appropriately, provide RNA which is equivalent to RNA from biopsies which are frozen immediately, but that multiple biopsy protocols may introduce additional complexities.

Biopsy, Needle↗
PubMed2006

The high viscosity encountered during freezing in glycerol solutions: effects on cryopreservation.

The objective of this study was to determine the viscosity of the residual unfrozen solution that cells are exposed to during freezing in the presence of glycerol and use this to interpret some key aspects of cryopreservation. The viscosity of the glycerol-water binary system exceeded 1000 cP at -40 degrees C, whilst the viscosity of the ternary system, glycerol-water-NaCl, exceeded 100,000 cP at -55 degrees C. The effect of these high viscosities on the diffusion of water at a constant temperature during freezing and during cooling at different linear rates has been estimated. At rates of cooling faster than 100 degrees C min(-1) the diffusion distance during freezing was calculated to be less than 15 microm. Validation of the diffusion calculations was confirmed by examination of the ultrastructure of the freeze concentrated matrix in samples prepared at a range of cooling rates. At a critical rate of cooling, water diffusion becomes limited by the high viscosity and two phenomena, of relevance to cryobiology, occur: (1) the composition of the freeze concentrated matrix around cells deviates from that of the equilibrium phase diagram; and (2) the osmotic loss of water from cells is restricted. These factors are of particular relevance to an understanding of the response of cells such as spermatozoa, red blood cells, and bacteria cooled rapidly with glycerol as cryoprotectant.

Cryopreservation↗
PubMed1996

An experimental study of the mechanical response of frozen biological tissues at cryogenic temperatures.

An experimental study of the mechanical response of frozen soft biological tissues to applied compressive stresses is presented. This study is related to the mechanical stresses that develop due to the contraction of frozen tissues in cryopreservation as well as in cryosurgical procedures. The main concept in this study is that the stresses associated with the constrained contraction of the frozen tissue, i.e., due to temperature variations within the frozen tissue, can be simulated by an external mechanical load which is applied to the frozen tissue while the tissue is maintained at a uniform temperature. A new apparatus for measuring compressive stresses and strains of frozen biological tissues in cryogenic temperature range is presented. A new technique for processing the fresh biological tissue into a cylindrical frozen sample for mechanical testing is introduced. Results of compression tests on rabbit liver, kidney, and brain are presented and are compared with available data from the literature on sea ice and single ice crystals. An unusual response of frozen biological tissues to compressive stress was observed: after the initial, roughly linear elastic portion there was a series of sudden stress drops at constant strain, each followed by a linear increase of stress with strain to the next drop. This phenomenon, which is attributed to the accumulation of microcracks, broadly resembles plastic deformation, and thus provides some support for simple mechanical models invoked in theoretical studies.

Animals↗
PubMed1995

Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes.

The aim of the reported study was to investigate the reproducibility of the single-cell gel electrophoresis (SCGE) assay in the determination of DNA single-strand breaks (SSBs) and to estimate the statistical requirements when the SCGE assay is used for the detection of genotoxicity in humans. In human peripheral mononuclear leukocytes (PMLs), we repeatedly measured the rate of SSBs after in vitro incubation of cells for 1 h at 4 degrees C in phosphate buffered saline (PBS, basal) or 10 microM or 50 microM H2O2 (induced). Intra-assay variation was determined from cryopreserved PMLs of a single donor. To assess intrasubject and intersubject variation, PMLs of ten healthy, nonsmoking subjects (aged 19-37 years) were tested 5-9 times. Cryopreserved cells revealed a mean coefficient of variation of 18% (PBS) and 7%-9% (H2O2). There were statistically significant differences between individuals in the rate of SSBs after incubation in PBS (P < 0.01), 10 microM H2O2 (P < 0.001), and 50 microM H2O2 (P < 0.001). The range of interindividual variability was 26% for basal and 12%-13% for induced SSBs, and the coefficient of intraindividual variation was 18%-72% (PBS) and 7%-23% (H2O2). Neither basal nor induced rates of DNA damage were related to gender or age. Estimates of the minimum detectable effects were based on these observed sources of variability (power 90%, level of significance 5%, assumed sample size 50). With two different groups, a difference of 31% in basal SSBs or 12% in induced SSBs would be detectable. Repeated measurement within one group could detect a difference of 26% in basal and 9% in induced SSBs. In summary, the SCGE assay appears to be suitable for the detection of single-strand breaks, e.g., in biomonitoring or environmental medicine, and the statistical requirements could be derived from our analysis of the sources of variability.

Adult↗
PubMed2000

Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B.bubalis) spermatozoa.

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy during capacitation and fertilization. Since cryopreservation procedures for Buffalo spermatozoa have not been adequately defined, the aim of the study was to improve the survival rate of buffalo (Bubalus bubalis) spermatozoa after cryopreservation by optimizing the timing for adding glycerol and by enriching the cryoprotectant extender with an energy source substrate. Semen was collected with an artificial vagina from 5 bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for viability, pooled and held at room temperature (28 degrees C) for 1 h. Then aliquots of pooled semen were subjected to dilution and equilibration in triplicate as follows: Experiment 1. Glycerol (3%) in a commercial extender was added to the semen at 28 degrees C and cooled to 5 degrees C for 1 h; then extender with 11% glycerol was added before further equilibration (initial glycerol addition; IGA) and the samples held at 5 degrees C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before freezing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5 degrees C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n= 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiments 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (IPA and LPA samples). The effect of addition time (initial vs late) of glycerol and pyruvate was evaluated by measuring sperm motility, progressively forward motility and viability. After freezing-thawing the percentage of motile spermatozoa was significantly higher (0.001<P<0.01) after a late addition of glycerol and pyruvate (LGA 5 and LPA 5). The optimizing of the timing of the glycerol addition and the presence in the extender of an energy source rendered a higher efficiency in thawed spermatozoa.

Animals↗