Sequestration of PDC‐109 Protein by Specific Antibodies and Egg Yolk Cryoprotects Bull Spermatozoa

PDC‐109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time‐ and concentration‐dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC‐109 either by anti‐PDC‐109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC‐109 protein was purified by applying two‐step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion‐exchange chromatography. After checking the Ab cross‐reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris‐glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC‐PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein‐sequestered‐semen (zona‐penetration assay) were evaluated. A significant (p < 0.05) improvement in post‐thaw SQPs as well as in non‐capacitated spermatozoa observed at pre‐freeze and post‐thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein‐mediated cryoinjury. From this study, it can be concluded that sequestration of PDC‐109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra‐low temperatures.     

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short‐term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short‐term storage and the swim‐up procedure. We used a standard protocol, for short‐term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro? (Minitüb GmbH, Tiefenbach, Germany). After each set‐up storage period, the motile fraction of sperm cells was selected by the swim‐up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non‐selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim‐up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species‐related differences.     

Effect of Swim up and Percoll Treatment on Viability and Acrosome Integrity of Frozen–thawed Bull Spermatozoa

The aim of this study was to investigate the effect of the swim up and Percoll methods to select frozen–thawed bull spermatozoa with high quality membrane and acrosomal integrity and final concentration. Semen samples from six Holstein–Friesian bulls were examined. The whole experiment was repeated three times. Before and after both treatments, spermatozoa were subjected to a double‐staining method and evaluated by brightfield light microscope using 40× dry, or 100× oil immersion objectives. The concentration of spermatozoa evaluated by haemocytometer was 8.8 × 10⁷/ml after thawing, and the percentage of live cells with intact acrosome was 45.8%. Both treatments significantly increased the proportion of live spermatozoa compared with no treatment, and the use of Percoll gradient resulted in a significantly higher percentage of living cells with an intact acrosome (88.2%) than the swim up method (69.4%). The concentration of spermatozoa after Percoll separation (9.3 × 10⁶/ml) was higher than that after the swim up method (5.8 × 10⁶/ml). These results indicate that spermatozoa with a higher viability and acrosome integrity can be obtained by Percoll separation than by the swim up method. Therefore the use of Percoll‐treated spermatozoa in IVF systems can be more expedient.     

Quantification of the DNA Difference,and Separation of X‐ and Y‐Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre‐determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome‐bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X‐ and Y‐chromosome and (iii) determine the DNA difference between X‐ and Y‐bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high‐speed cell sorter (SX‐MoFlo^®). H33342 staining concentrations of 36, 54, 72 or 90 μm did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X‐ and Y‐enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX‐MoFlo^®), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin‐like Growth Factor I,Insulin and Membrane Integrity of Bull Spermatozoa

This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin‐like growth factor I (IGF‐I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre‐freezing and post‐thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment‐by‐collection day on plasma concentrations of IGF‐I and insulin was not significant. However, mean plasma concentrations of IGF‐I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment‐by‐collection day on motility of spermatozoa was similar (p > 0.05) at pre‐freezing and post‐thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post‐thawing stage was higher (p < 0.05) in rbST‐treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF‐I and insulin, however, it did improve post‐thaw sperm membrane integrity.     

Localization of CD9 Molecule on Bull Spermatozoa: Its Involvement in the Sperm–Egg Interaction

Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA‐50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen‐thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti‐CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.     

In vitro Fertilizing Capacity of Frozen-thawed Bull Spermatozoa Selected by Single-layer (Glycidoxypropyltrimethoxysilane) Silane-coated Silica Colloidal Centrifugation

Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll™ (C2). Sperm recovery surpassed 50% for both C1–C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p < 0.05). In T, the mean (±SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 ± 23.3%, 24.5 ± 14.3% and 94.6 ± 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.     

Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen–Thawed Stallion Spermatozoa

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E™). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen–thawed semen samples.     

Facilitation of an Exotic Grass Through Nitrogen Enrichment by an Exotic Legume

Invasive species control requires understanding the mechanisms behind their establishment and their interactions with other species. One potential ecosystem alteration influencing the establishment and spread of invasive species is anthropogenic nitrogen enrichment, from sources like introduced or invasive nitrogen (N)-fixing legumes, which can alter competition between native, non-native, and invasive plants. Kentucky bluegrass (Poa pratensis) and N-fixing yellow sweet clover (Melilotus officinalis) are exotic to the Great Plains and are currently invading and degrading native rangelands by altering ecosystem processes and displacing native plants. Therefore, we investigated how N enrichment from yellow sweet clover affects the aboveground biomass production of Kentucky bluegrass and western wheatgrass (Pascopyrum smithii), a native cool-season grass, the ranges of which overlap in the northern Great Plains. In a controlled greenhouse environment, we conditioned experimental pots by growing yellow sweet clover and terminating each plant after 8 wk. Conditioned soils contained ≈ 340% more plant-available N than untreated soils 2 wk after yellow sweet clover death. We then grew Kentucky bluegrass and western wheatgrass transplant seedlings in interspecific and intraspecific pairs in pots conditioned either with or without yellow sweet clover for 12 wk. Aboveground biomass production of both Kentucky bluegrass and western wheatgrass grown in interspecific and intraspecific pairs increased in conditioned soils. However, when grown together in conditioned pots, the increase in Kentucky bluegrass biomass relative to untreated pots (520%) was double that of the increase in western wheatgrass biomass (260%). Our results reveal that Kentucky bluegrass can use increased soil N to produce proportionally more aboveground biomass than western wheatgrass, a native grass competitor. Thus, our results suggest yellow sweet clover and other sources of N enrichment may facilitate the invasion of Kentucky bluegrass.     

Development of Y‐chromosome‐Specific SCAR Markers Conserved in Taurine,Zebu and Bubaline Cattle

Sex pre‐selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR‐specific primers are derived from the few single‐copy Y‐chromosome‐specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male‐specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male‐specific markers (OPC16₃₂₃ and OPF10₁₁₆₈) by means of RAPD. These markers were successfully converted into SCARs (OPC16₇₂₆ and OPF10₉₈₄) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16₃₂₃ marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16₃₂₃ is also highly similar to a bubaline Y‐chromosome‐specific sequence. The primers derived from the two Y‐chromosome‐specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

Cell Subpopulation‐related Volumetric Parameters: a Complementary Tool of the Modified Hypo‐osmotic Swelling Test on Model of Boar Spermatozoa

It is a general property of the intact animal cell to swell rapidly in response to hypo‐osmotic conditions. The modified hypo‐osmotic swelling test (HOS‐test) is an indicative test to evaluate the integrity of the plasma membrane by means of an electronic cell counter, based on the relative increase of the cell volume in response to hypo‐osmotic conditions. In this study the relationships between the osmotically induced changes of the cell volume of boar spermatozoa as determined by cell counter and the integrity of the membrane as determined by propidium iodide staining (PI) were studied. Boar sperm cell volume distributions were measured under iso‐osmotic (300 mosmolar) conditions and after a hypo‐osmotic stress (150 mosmolar). The relative volume shift of mean and modal volume were calculated as a proportion coefficient of modal and mean values of the cell volume distributions by transition from iso‐osmotic to hypo‐osmotic conditions. The volumetric parameters related to the different cell subpopulations were derived from the different peaks of cell volume distributions. PI‐staining techniques were used for comparison. The values of the volume shift and of derived percentages of the osmotically inactive cells were correlated negatively and positively, respectively (p < 0.05) with the percentage of the PI‐stained cells. This correlation indicates that a relationship exists between membrane functions of the different cell compartments (sperm head and tail) due to the circumstance that the increase of the cell volume in the HOS‐test is associated with the morphological changes in the tail and the PI‐staining is associated with the membrane integrity and permeability of the head region. The advantage of computer‐assisted volume measurement is that a large number of cells (5000–50 000 spermatozoa) can be measured and evaluated during one procedure and in a very short time. The relative volume shift is a quantitative continuous parameter characterizing the osmotic reactivity and membrane functional competence of a cell population and of subpopulations within one ejaculate. This parameter could be useful to evaluate membrane functional competence rapidly and sensitively.     

Post-thaw Survival and Longevity of Bull Spermatozoa Frozen with an Egg Yolk-based or Two Egg Yolk-free Extenders after an Equilibration Period of 18 h

The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed and Biociphos Plus as compared with the Tris-egg yolk based diluent Biladyl, for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mul aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl results in higher sperm survival and longevity than the use of Andromed or Biociphos Plus.