Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.     

Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris--egg yolk freezing media, TEST and TTE, which have been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa from four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes, 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts, respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species.     

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.     

The cryopreservation of spermatozoa of the burbot,Lota lota (Gadidae,Teleostei)

The cryopreservation of spermatozoa of a teleost fish, the burbot, Lota lota (Gadidae) was investigated. Cryopreserved semen had the highest motility rate (46.6+/-8.0%, fresh semen control 86.5+/-8.2%) and fertility (78.1+/-2.7% embryo survival in hatching stage, fresh semen control 82.2+/-2.9%) when 10% methanol, 1.5% glucose and 7% hen egg yolk were used as cryoprotectants. Freezing was performed in 0.5-ml straws in the vapour of liquid nitrogen at 1cm above the level of liquid nitrogen and thawing in water at 25 degrees C for 20s. For optimal fertilization cryopreserved semen was first mixed with the eggs and then 25 or 50 mmol/L NaCl solution (pH 8.5) was added at a ratio of 1:24 (semen:saline solution). Under these conditions fertilization ratios in the range of fresh semen control were obtained at minimal sperm to egg ratios of 1.7 x 10(6):1. Fertilization with cryopreserved semen had no influence on the embryonic development, as the ratio of embryos which stopped development and the ratio of embryonic malformations were similar to fresh semen.     

Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

The study of DNA integrity in somatic cells and spermatozoa by single cell gel electrophoresis with silver staining

In present work we studied DNA damage in human and bovine lymphocytes and spermatozoa by means of single cell gel electrophoresis followed by silver staining. The spontaneous frequency of DNA damage estimated manually in spermatozoa from healthy donors did not exceed 9% (on average -- 4.8 +/- 1.2%). The frequency of DNA damages in bull sperm after short (less than a year) and long period (more than 20 years) of cryopreservation was assessed as 3.1 +/- 0.9 and 4.3 +/- 0.5%, correspondingly. The comparative estimation of DNA damages in lymphocytes followed by silver staining is a valuable tool to estimate DNA damage in populations of somatic and reproductive cells.     

Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm

Occasionally, boar semen must be shipped to another location for cryopreservation. We increased the initial holding time for the cooling of extended semen at 15 degrees C from 3 to 24 h to determine the effects on sperm characteristics and fertility. Thirty-one gilts and sows were inseminated once with subsequently cryopreserved and thawed semen. Increasing the holding time from 3 to 24 h had no significant effect on pregnancy rate 23 days after AI with frozen-thawed semen (64.5%) but decreased (P<0.05) embryo number from 15 to 9 and recovered embryos as fraction of CL from 73 to 47%. While the longer holding time at 15 degrees C did decrease potential litter size, the loss incurred was not too great to preclude the incorporation of a longer holding time into the cryopreservation protocol. An experiment was conducted to test the hypothesis that processing and freeze-thawing of boar semen would induce phospholipid scrambling in the plasma membrane similar to that evoked by incubation in bicarbonate-containing media. Merocyanine staining after incubation in the presence and absence of bicarbonate indicated that changes in plasma membrane phospholipid scrambling of processed and cryopreserved sperm differed from those in fresh semen undergoing bicarbonate-induced capacitation. The level of Annexin-V binding in boar spermatozoa increased from 1.6% in live spermatozoa in fresh semen to 18.7% in cryopreserved sperm. Apoptosis is unlikely to operate in mature spermatozoa. Apoptotic morphology in ejaculated spermatozoa is probably a result of incomplete deletion of apoptotic spermatocytes during spermatogenesis. Increased Annexin-V binding in thawed spermatozoa probably results from plasma membrane damage incurred during freezing and thawing.     

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 +/- 1.6 to 30.0 +/- 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.     

In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products.

Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitro handling of stallion sperm may be isolated from OEC secretory products. Experiment 1 compared first service conception (FSC) rates resulting from the use of cryopreserved sperm of seven stallions, with sperm function in co-culture such as attachment to OEC and subsequent survival time. Stallions were grouped by cumulative FSC rates observed over three seasons as having average (44 +/- 3%) or high (65 +/- 2%) fertility over a total of 217 first services (31 +/- 9 per stallion). Samples from stallions in the high fertility group had more (P = 0.04) sperm attached to OEC and longer subsequent sperm survival in co-culture (P = 0.05) as compared with those from the average fertility group. FSC rates correlated with numbers of sperm attaching to OEC and their survival time in co-culture (r > or = 0.71). In Experiment 2, the function of cryopreserved stallion sperm was evaluated in culture with OEC secretory products from three different sources. After 5 h of culture, sperm incubated with medium conditioned by bovine OEC which had been 'bioactivated' (e.g. previously exposed to sperm in culture) were found to be more (P < or = 0.05) motile and capacitated as compared to sperm in basal TALP medium alone. Sperm in this conditioned medium also survived longer (P = 0.05; 27 +/- 5 h vs. 17 +/- 4 h) than did those in control medium.