Complex interactions between nicotine and resveratrol in the Drosophila melanogaster wing spot test.

Nicotine (NIC) and resveratrol (RES) are chemicals in tobacco and wine, respectively, that are widely consumed concurrently worldwide. NIC is an alkaloid known to be toxic, addictive and to produce oxidative stress, while RES is thought of as an antioxidant with putative health benefits. Oxidative stress can induce genotoxic damage, yet few studies have examined whether NIC is genotoxic in vivo. In vitro studies have shown that RES can ameliorate deleterious effects of NIC. However, RES has been reported to have both antioxidant and pro-oxidant effects, and an in vivo study reported that 0.011 mM RES was genotoxic. We used the Drosophila melanogaster wing spot test to determine whether NIC and RES, first individually and then in combination, were genotoxic and/or altered the cell division. We hypothesized that RES would modulate NIC's effects. NIC was genotoxic in the standard (ST) cross in a concentration-independent manner, but not genotoxic in the high bioactivation (HB) cross. RES was not genotoxic in either the ST or HB cross at the concentrations tested. We discovered a complex interaction between NIC and RES. Depending on concentration, RES was protective of NIC's genotoxic damage, RES had no interaction with NIC, or RES had an additive or synergistic effect, increasing NIC's genotoxic damage. Most NIC, RES, and NIC/RES combinations tested altered the cell division in the ST and HB crosses. Because we used the ST and HB crosses, we demonstrated that genotoxicity and cell division alterations were modulated by the xenobiotic metabolism. These results provide evidence of NIC's genotoxicity in vivo at specific concentrations. Moreover, NIC's genotoxicity can be modulated by its interaction with RES in a complex manner, in which their interaction can lead to either increasing NIC's damage or protecting against it.

Lycopene, resveratrol, vitamin C and FeSO4 increase damage produced by pro-oxidant carcinogen 4-nitroquinoline-1-oxide in Drosophila melanogaster: Xenobiotic metabolism implications.

4-nitroquinoline-1-oxide (4-NQO) is a pro-oxidant carcinogen bioactivated by xenobiotic metabolism (XM). We investigated if antioxidants lycopene [0.45, 0.9, 1.8 µM], resveratrol [11, 43, 172 µM], and vitamin C [5.6 mM] added or not with FeSO4 [0.06 mM], modulate the genotoxicity of 4-NQO [2 mM] with the Drosophila wing spot test standard (ST) and high bioactivation (HB) crosses, with inducible and high levels of cytochromes P450, respectively. The genotoxicity of 4-NQO was higher when dissolved in an ethanol - acetone mixture. The antioxidants did not protect against 4-NQO in any of both crosses. In the ST cross, resveratrol [11 µM], vitamin C and FeSO4 resulted in genotoxicity; the three antioxidants and FeSO4 increased the damage of 4-NQO. In the HB cross, none of the antioxidants, neither FeSO4, were genotoxic. Only resveratrol [172 µM] + 4-NQO increased the genotoxic activity in both crosses. We concluded that the effects of the antioxidants, FeSO4 and the modulation of 4-NQO were the result of the difference of Cyp450s levels, between the ST and HB crosses. We propose that the basal levels of the XM's enzymes in the ST cross interacted with a putative pro-oxidant activity of the compounds added to the pro-oxidant effects of 4-NQO.

Detection of Bovine Viral Diarrhoea Virus 2 as the Cause of Abortion Outbreaks on Commercial Sheep Flocks.

Outbreaks of abortions and lambs born with nervous clinical signs and/or congenital malformations affected different sheep farms in Spain. Initial diagnosis of 'border disease-like' was established, based on clinical signs, serology and/or RNA detection by a pan-pestivirus RT-PCR. However, further investigation using immunohistochemical and molecular techniques identified BVDV-2b as the aetiological agent.

Acanthoparyphium sp. and other metazoan symbionts of the American oyster, Crassostrea virginica, from South Texas.

In this study, we investigated oysters, Crassostrea virginica , from Laguna Madre in South Texas, where a 45-yr old study recorded metacercarial infections of the echinostomatid trematode, Acanthoparyphium spinulosum , an Asian relative of which, Acanthoparyphium tyosenense, has been associated with human infections via the ingestion of raw mollusks. In an effort to examine the base-line infection parameters of Acanthoparyphium sp. in oysters, we examined the effect of distance from the shoreline, which is the habitat of the first intermediate host snail, Cerithidea pliculosa, as well as temporal changes in oyster infection levels, by conducting quarterly collections of oysters during a year. We found that almost all oysters (prevalence = 97.8-100%) were infected regardless of distance to the shoreline and season. However, the abundance of metacercariae was significantly higher close to the shoreline, while no significant temporal changes could be detected. In addition to the echinostomatid, we found a high abundance of the metacestode Tylocephalum sp. and the presence of 4 other metazoan parasites. None of the infections seemed to incur significant tissue damage to the oysters. Our study shows that at least locally, recreational harvesters of oysters may be exposed to Acanthoparyphium sp. Future studies should examine oysters from snail host habitats in the Gulf of Mexico, and the potential zoonotic risk of Acanthoparyphium sp. should be evaluated using experimental infections of animal models.

Interactions of sulforaphane and dimethyl sulfoxide with methyl methanesulfonate, urethane, 4-nitroquinoline-1-oxide and hydrogen peroxide in the Drosophila melanogaster wing spot test.

Sulforaphane (SF) is an isothiocyanate present in Brassicaceae, vegetables that induce the detoxification of electrophiles and reactive oxygen species. SF has been correlated with chemoprevention mechanisms against degenerative diseases. We tested if the SF had an effect against methyl methanesulfonate (MMS), urethane (URE), 4-NQO and H(2)O(2). SF (>95% purity, 0.14, 0.28, 0.56 mM) was diluted in a DMSO/Tw80/EtOH mixture (DTE) corresponding to 25, 50, 100% of lyophilized broccoli. The SF treatment (0.14 mM) was positive for small spots in the ST cross and negative in the HB cross. In the HB cross, SF (0.28 mM) was genotoxic. In the ST cross, the SF treatments showed a tendency to reduce the genotoxic damage caused by MMS, which could be explained by the radical scavenging action of the DTE mixture. In the ST cross, the frequency of small spots in the SF 0.14 mM/URE treatment was similar to that of Water/URE, which can be explained by a DTE and SF scavenger action. In both crosses, the results for the direct oxidants, 4-NQO and H(2)O(2), were different and must be related to differential modulation of CYPs expression and the SF and DTE scavenger properties.

The generation of live offspring from vitrified oocytes.

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.

Sulforaphane modulates the expression of Cyp6a2 and Cyp6g1 in larvae of the ST and HB crosses of the Drosophila wing spot test and is genotoxic in the ST cross.

Constitutive overexpression of Cyp6g1 and Cyp6a2 genes in DDT-resistant line Oregon-flare of the Drosophila melanogaster wing spot test (SMART) has been reported. Cyp6g1 and Cyp6a2 expression levels were compared against the ß-actin gene in the standard (ST) and high bioactivation (HB) crosses of the Somatic Mutation and Recombination test (SMART) treated with sulforaphane or phenobarbital as the control inductor. The CYP450s' enzymatic activity was determined by overall NADH consumption. The expression levels of both genes and the CYP450s activity was higher in the HB cross. The Cyp6g1 levels were higher than those of Cyp6a2 in both crosses, but lower than the expression of ß-actin. Sulforaphane decreased Cyp6g1 in the HB cross and increased it in the ST cross; Cyp6a2 expression was inhibited in the ST cross. Sulforaphane resulted mutagenic in the ST cross, which could be related to the inhibition of Cyp6a2. Phenobarbital did not modify the Cyp6g1 levels but increased the Cyp6a2 and CYP450s basal activity. Although the transcript levels were always higher in the HB cross than in the ST, the expression of Cyp6a2 and Cyp6g1 was not constitutive and was independent one from the other. Sulforaphane modulated both genes in a differential way in each cross and, in contrast to its putative protective effect, it resulted to be mutagenic.

Glioma pathogenesis-related 1-like 1 is testis enriched, dynamically modified, and redistributed during male germ cell maturation and has a potential role in sperm-oocyte binding.

The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization.

Activin C antagonizes activin A in vitro and overexpression leads to pathologies in vivo.

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-beta(C) subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-beta(C). Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LbetaT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-beta promoter. Transgenic mice that overexpress activin-betaC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-beta(C) antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-betaC immunoreactivity. This study provides evidence that activin-beta(C) is an antagonist of activin A and supplies an impetus to examine its role in development and disease.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

Complex expression patterns support potential roles for maternally derived activins in the establishment of pregnancy in mouse.

Maternal-fetal communications are critical for the establishment of pregnancy. Embryonic growth and differentiation factors produced by the oviduct and uterus play essential roles during the pre- and early post-implantation phases. Although several studies indicate roles for activin in embryonic development, gene-knockout studies have failed to identify a critical role in mammalian embryogenesis. We hypothesized that activin is produced by maternal tissues during the establishment of pregnancy, and thus maternally derived activin could compensate for the absence of embryonic activin in null homozygotes during critical developmental stages. We investigated the expression of inhibin alpha, activin betaA, and betaB subunits in the mouse oviduct and uterus during the estrous cycle and early pregnancy, and in the early conceptus. Inhibin alpha subunit was weakly expressed, while activin betaA and betaB subunits were strongly expressed in oviduct and uterus at estrous, and dramatically upregulated in the uterus on each day of pregnancy between days 3.5 and 8.5 post coitum. Prior to implantation, activin betaA and betaB subunits were immunolocalized to oviductal and uterine epithelial cells; following implantation they were expressed in the stroma, in a wave preceding decidualization. Later in pregnancy, activin betaA and betaB subunits were present in decidua basalis, trophoblast giant cells, and labyrinth zone of the developing placenta. Expression of activin betaA subunit was also detected in blastocysts and early post-implantation embryos. These data are consistent with a role for maternally derived activins in the support of the pre-implantation embryo, and during gastrulation and embryogenesis.

Ram sperm motility after intermittent scrotal insulation evaluated by manual and computer-assisted methods.

AIM: To study whether additional measurements of motility characteristics of spermatozoa by computer assisted semen analysis (CASA) were more sensitive indicators of reduced semen quality than estimates of percentages of motile, rapid or progressive cells. METHODS: Intermittent scrotal insulation was applied to 6 rams for 16 h per day for 21 days or to 2 of these for 12 h per day for 28 days in the following year. Semen was collected and evaluated by CASA immediately and either frozen or stored at 30 degrees Celsius or 5 degrees Celsius before re-evaluation. RESULTS: Intermittent scrotal insulation caused falls in the percentage of motile, progressive and rapid sperm, as did freezing-thawing and storage at 30 degrees Celsius or 5 degrees Celsius. Motility characteristics (amplitude of lateral head displacement, mean path velocity, mean progressive velocity and curvilinear velocity), as determined by CASA fell only when the percentage of motile sperm was already reduced. Freezing and thawing or liquid storage of the semen from insulated rams caused a greater fall in the percentage of motile and rapid sperm than control semen, but only affected the motility characteristics when the percentage of motile sperm was already reduced. CONCLUSION: Intermittent scrotal insulation affected not only the motility of the freshly collected sperm, but also their ability to withstand the additional stress of storage. The additional data on motility characteristics obtained by CASA appeared to be no more a sensitive indicator than the percentage of motile cells of reductions in semen quality.

FGFR-1 [corrected] signaling is involved in spermiogenesis and sperm capacitation.

Cloning of the fibroblast growth factor receptor (FGFR) adaptor Snt-2 cDNA and the identification of FGFR-1 protein in association with sperm tails, suggested that FGFR-1 signaling was involved in either sperm tail development or function. This hypothesis was tested by the creation of transgenic mice that specifically expressed a dominant-negative variant of FGFR-1 in male haploid germ cells. Mating of transgenic mice showed a significant reduction in pups per litter compared with wild-type littermates. Further analysis demonstrated that this subfertility was driven by a combination of reduced daily sperm output and a severely compromised ability of those sperm that were produced to undergo capacitation prior to fertilization. An analysis of key signal transduction proteins indicated that FGFR-1 is functional on wild-type sperm and probably signals via the phosphatidylinositol 3-kinase pathway. FGFR-1 activation also resulted in the downstream suppression of mitogen activated protein kinase signaling. These data demonstrate the FGFR-1 is required for quantitatively and qualitatively normal spermatogenesis and has a key role in the regulation of the global tyrosine phosphorylation events associated with sperm capacitation.

A repository of ENU mutant mouse lines and their potential for male fertility research.

Many of the proteins and their encoding genes involved in spermatogenesis are unknown, making the specific diagnosis and treatment of infertility in males difficult and highlighting the importance of identifying new genes that are involved in spermatogenesis. Through genome-wide chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and a three-generation breeding scheme to isolate recessive mutations, we have identified mouse lines with a range of abnormalities relevant to human male fertility. Abnormal phenotypes included hypospermatogenesis, Sertoli cell-only (SCO) seminiferous tubules, germ-cell arrest and abnormal spermiogenesis and were accompanied, in some, with abnormal serum levels of reproductive hormones. In total, from 65 mouse lines, 14 showed a reproductive phenotype consistent with a recessive mutation. This study shows that it is feasible to use ENU mutagenesis as an effective and rapid means of generating mouse models relevant to furthering our understanding of human male infertility. Spermatozoa and genomic DNA from all mouse lines, including those with abnormal reproductive tract parameters, have been cryopreserved for the regeneration of lines as required. This repository will form a valuable resource for the identification and analysis of key regulators of multiple aspects of male fertility.

Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains.

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications.

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm.

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.

Fertility and its relationship to motility characteristics of spermatozoa in ewes after cervical, transcervical, and intrauterine insemination with frozen-thawed ram semen.

The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.

Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm.

The effect of the compatible solutes proline, glycine betaine and trehalose in Tris-based diluents at varying pH, concentrations of egg yolk or glycerol on the post-thaw motility characteristics and fertility of ram sperm was examined. In addition, the amino acid glycine was compared with proline, glycine betaine and a standard Tris-based diluent. Post-thaw motility was assessed using a Hamilton-Thorn motility analyser. In the presence of glycerol and egg yolk, proline and glycine betaine improved the post-thaw motility characteristics of ram sperm. Regardless of the pH of the diluent at which semen was frozen, the percentage of motile sperm was higher when frozen in the presence of proline or glycine betaine than in their absence, whereas proline and glycine betaine only improved the progressive and rapid percentages of sperm for semen frozen in diluents at pH lower than 7.0. When semen was frozen in the absence of egg yolk or glycerol all the motility characteristics were reduced. Increasing the concentration of egg yolk in the diluent from 5% to 10, 15 or 20% had no effect on the post-thaw motility of sperm. The addition of 27 mM of proline or glycine betaine to the diluent also improved post-thaw motility. However, at a concentration of 81 mM, proline and glycine betaine had a detrimental effect on the percentage of motile sperm. Trehalose had no effect on the motility of sperm frozen in glycerol-containing diluents, but motility was lower after cryopreservation in glycine than in Tris-, proline- or glycine betaine-based diluents. There were no differences in the fertility of sperm frozen in Tris-, proline or glycine betaine diluents after cervical or laparoscopic insemination of ewes.

Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa.

The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.

Distribution of variance associated with measurement of post-thaw function in ram sperm.

Post-thaw characteristics of ram semen frozen as pellets were assessed using biochemically (amidase activity) or motility-based (Hamilton Thorn Motility Analyzer) techniques. The total variation associated with each semen characteristic measured was partitioned between rams (5), ejaculates within rams (5), pellets within ejaculates (5) and within pellets (2). A variety of variance distributions were observed for the characteristics measured. Of the 18 post-thaw characteristics examined, 10 had > 50% of variance distributed between within-ejaculate components. This has important implications for the way in which such measurements may be used in post-thaw semen analysis.