Semen Evaluation Research Articles

Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat.

The current study aimed to evaluate the relationship between specific seminal plasma components - cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations - and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Thirteen toms, 2-5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at -20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats (P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

Cytosolic phospholipase A2 and F2 isoprostanes are involved in semen quality and human infertility-A study on leucocytospermia, varicocele and idiopathic infertility.

Phospholipase A2 (PLA2 ) is involved in eicosanoid release, and F2 -isoprostanes (F2 -IsoPs), as free radical-generated eicosanoids released by PLA2 , are indicators of oxidative stress in different human conditions. This study investigated the interplay between cytosolic PLA2 (cPLA2 ), F2 -IsoPs and sperm features in male infertility, when the involvement of oxidative stress has been reported. Semen evaluation was performed following WHO guidelines, sperm ultrastructure was detected by transmission electron microscopy indicating a fertility index, and the percentages of sperm immaturity, apoptosis and necrosis. In sperm cells and seminal plasma, cPLA2 levels were determined by immunological method, whereas F2 -IsoPs by mass spectrometry. Sperm concentration, morphology, vitality and fertility index values were significantly lower in infertile groups compared with fertile men. An increase in sperm apoptosis and necrosis (p<.01), apoptosis (p<.01) and immaturity (p<.001) was detected in leucocytospermia, idiopathic infertility and varicocele, respectively. Seminal cPLA2 showed the highest value in varicocele group (p<.05), whereas seminal F2 -IsoPs increased in varicocele (p<.001) and leucocytospermia (p<.05) groups. In the whole population, F2 -IsoP and cPLA2 levels were positively correlated (p<.05). On the contrary, F2 -IsoPs and cPLA2 were not significantly different when investigated in sperm cells. Our data indicate that fatty acid oxidation/metabolism plays a role in different male reproductive pathological conditions.

Effect of temperature and time after collection on buck sperm quality

BackgroundDifferent parameters are assessed as part of the semen analysis but a standard protocol for evaluation of goat semen is still missing. The aim of this study was to analyse two different factors affecting buck sperm quality in the post-collection period prior to adding the extender. Here we examined the effects of two handling temperatures (20 °C, 37 °C) and various examination time points (3–30 min) after semen collection.ResultsExamination time point had a significant influence on raw sperm viability (p < 0.05), motility (p < 0.05) and on semen pH (p < 0.05). The two different handling temperatures had no significant effect on sperm viability (p > 0.05), motility (p > 0.05), with the exception of fast moving sperm (p = 0.04), or on semen pH (p > 0.05).ConclusionExamination time point was identified as factor strongly influencing raw peacock buck semen after collection. Raw goat semen can tolerate room temperatures for at least 10 min without impacting overall semen quality. In order to obtain comparable results, semen samples should always be examined within 10 min after collection.

A non-invasive method for semen collection and evaluation in small and median size lizards.

The aim of this study was to develop a non-invasive procedure to stimulate ejaculation in median and small lizards for semen collection. After semen collection, we applied a battery of tests to assess the motility (wave and progressive), viability (eosin-nigrosin stain), morphology (normal/abnormal), sperm concentration and ejaculate number, seminal volume and colour. We obtained this fluid from all males of the four species of sceloporine lizards (n=30) and one species of Mexican horned lizards (n=7). We found that semen from all males had a liquid-like consistency and a milky-white appearance. Ejaculate volume and sperm concentration varied among the males and the species studied. We also observed that although the mobility, viability and normal sperm morphology showed a wide variability, their average value in each species was high. We conclude that "genital papilla pressure" was an efficient and non-invasive semen obtaining method for small- and medium-sized lizards that does not imply damage to donors and could be used in other lizard species.

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English

Evaluation of the effect of laboratory methods on semen analysis and breeding soundness examination (BSE) classification in stallions

The stallion Breeding Soundness Examination (BSE), as proposed by the Society for Theriogenology, recommends that a stallion produce a minimum of one billion progressively motile, morphologically normal sperm (PMMNS) in the second of two ejaculates collected 1 h apart to be classified as a Satisfactory Prospective Breeder. With this in mind, the first objective of this study was to determine if the classification outcome of the traditional BSE differs depending on the methods used to evaluate sperm motility, morphology and concentration. We hypothesized that application of Computer Assisted Sperm Motion Analysis (CASA) and Differential Interference Contrast (DIC) microscopy to stallion semen evaluation would yield a more conservative estimate of the number of PMMNS. If this hypothesis is correct, then the use of CASA and DIC microscopy for semen evaluation would result in significantly fewer stallions meeting the historical standards for classification as a Satisfactory Prospective Breeder. Additionally, we determined whether the use of these modern technologies resulted in more accurate prediction of the actual fertility of a stallion compared to the use of more traditional technologies. Our results support the hypothesis that modern semen analysis techniques (including CASA and DIC microscopy) result in more conservative estimates of the number of PMMNS when compared to standard semen analysis techniques. As a result, the choice of methods used for semen analysis may impact the outcome of the traditional BSE. However, none of the methodologies used in this study reliably predicted different levels of fertility among this group of moderately to highly fertile stallions within the context of the traditional BSE. Additionally, the only individual semen measure that was significantly correlated with fertility was the percentage of morphologically normal sperm as determined using DIC microscopy. These results caution against strict use of the traditional ‘cutoff’ of 1 billion PMMNS for classification of breeding potential, particularly when attempting to differentiate between moderately and highly fertile stallions and regardless of the laboratory methods employed for semen analysis.

Evaluation of Scrotal Temperature in Different Testicular Shapes by Infrared Thermography in Braford Bulls

Background: The mechanisms of testicular thermoregulation may influence the blood flow provided by the testicular artery, where the proper blood supply to the testicle is crucial for promotingspermatogenesis and reproductive function in bulls. The size and shape of the testicles are determined by genetic mechanisms and environmental effects. A better understanding of the relationships between the anatomical characteristics of the testicles and scrotum can support a better reproductive assessment. The purpose of the current study was to evaluate the testicular temperature of different scrotal shapes using infrared thermography in bulls.Materials, Methods &amp; Results: We evaluated 132 Braford bulls with an average age of 24 months. The evaluation of breeding bull semen was performed prior to the beginning of the experiment. Then, animals were selected on the basis of the size of their testes, which was determined by dividing the average width by the average length. The scrotal circumference was measured with a millimeter tape positioned around the largest circumference. Testicular and ocular temperature measurements and analysis were conducted using an infrared thermal camera, Flir T440 with emissivity of 0.98 and thermal sensitivity of 0.05°C. Testicular scrotum temperature and testicular shape were analyzed with one-way ANOVA using Minitab 16, and values of P &lt; 0.05 were considered statistically significant. We observed that 67.42% of testicle shapes were long-oval, and 32.58% were long-moderate. The testicular temperature was higher in bulls with the long-moderate shape compared to those with the long-oval shape (P &lt; 0.05). The mean length was higher in long-moderate shaped testicles compared to those of the long-oval shape (P &lt; 0.01). There was no significant differences in rectal and ocular temperatures or in scrotal circumference between bulls with long-moderate and long-oval shapes (P &gt; 0.05). In addition, the mean width was lower in testicles of long-moderate shape compared to those of the long-oval scrotal format (P &lt; 0.01).Discussion: The results obtained showed that Braford bulls with the long-moderate testicular shape have a higher testicular temperature to maintain proper thermoregulation. The present study demonstrated that IRT can be used to evaluate the testicular temperature in animals with different scrotal conformations. In this study, Braford bulls showed lower length and width values for animals having long-moderate (9.21 and 5.22, respectively) and long-oval formats (8.56 and 5.56, respectively). In contrast, previous reports examining Nellore bulls between the ages of 17-20 months found a predominance of the long oval shape followed by the long-moderate shape, which indicates a change in testicular shape as age progresses, resulting in a rounder testicular shape. Perhaps other factors, such as the external cremaster muscle and tunica dartos, cause the testicles to be retracted towards the body at lower temperatures while at high temperatures, relaxation occurs. The prevailing testicular shape in Braford animals with a mean age of 24 months was the long-moderate shape. Thus, testicles with a larger surface area will have lower temperatures because they can dissipate heat more easily than testicles with lower surface area. The results suggest that the long-moderate scrotum format may influence the testicular temperature in mature Braford bulls.

Ameliorative Effect of Ascorbic Acid and Glutathione in Combating the Cryoinjuries During Cryopreservation of Exotic Jack Semen

The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L), GSH (2.5 mM), or combination of both (AA 0.9 g/L + GSH 2.5 mM), and another aliquot was kept as control without adding the antioxidants. The jack semen underwent cryodamage, which was evident by the observation of significant (P < .05) decline in the seminal quantitative parameters at various stages of cryopreservation process. Prefreeze and postthaw semen evaluation revealed that the values of plasma membrane, acrosome integrity, and chromatin integrity were found to be significantly higher (P < .01) in the group of samples supplemented with the combination (0.9 g/L AA +2.5 mM GSH) than AA- and GSH-alone or control groups. Supplementation of antioxidants to the freezing extender improved jack prefreeze and postthaw semen quality with the superiority of GSH over AA alone. From the present study, it was inferred that, exotic jack spermatozoa are susceptible to injuries because of cryopreservation, but these cryo-induced damage can be ameliorated significantly (P < .05) with the use of antioxidants and contribute to the improvement of semen cryopreservation procedures.

Maximizing semen extraction from sanitary pads by chemical and shredding treatments

Evidence of sexual aggression may be obtained from superabsorbent polymer (SAP) sanitary pads, which are used by forensic laboratories for semen evaluation. Semen can be extracted from their upper layers, which are free of SAPs. However, our previous results showed a need to optimize the protocol for semen analysis by considering its extraction from the lower core, often composed of sodium polyacrylate SAPs. SAPs generate a hydrogel, which traps the cellular components, hindering the possibility of obtaining cells and hence their genetic material. Simple filtration has been tried previously, but further maximization by application of a treatment has never been attempted. In this paper, we compare both chemical and physical shredding treatments for maximizing gel-trapped sperm and male cell DNA recaptures from hygienic superabsorbent substrates in sanitary pads, panty-liners or diapers. Our findings suggest that the lower core should be treated to induce a dewaterisation of the SAP hydrogels in order to maximize the extraction of bodily fluids.

Preservation of goat semen at 5°C with emphasis on its freezability and the impact of melatonin

Evaluation of goat semen, extended in Cornell University 16 extender, was used to study the effects of melatonin of various concentrations on sperm motility (SM) %, alive sperm (AS) % and sperm abnormalities (SA) % of extended goat semen stored at 5°C for seven days were studied.

Sperm morphology in relation to semen cation concentration in Yankasa rams

The morphological appearance of sperm cells is a major criterion in semen evaluation. The optimal production of semen of high biological value is influenced by numerous factors. Therefore, a study was conducted to evaluate the relationship between sperm morphological characteristics and semen cation concentrations in 62 rams of Yankasa breed of sheep. Semen samples were collected from each animal on weekly basis for 52 weeks with electro ejaculator. The sperm morphological characteristics examined were: detached mid-piece and tail (DMT), detached head (DH), mid-piece droplet (MPD, coiled and bent tail (CBT) and acrosomal abnormality (ACR). The semen cation parameters measured were: sodium + + 2+ ion (Na ), potassium ion (K ) and calcium ion (Ca ). Pearson correlation matrix of SAS was used to determine the relationships among and between the measured characteristics. The result of the study showed that correlations among the sperm morphological characteristics were low and not significant except that between DH and DMTwhich was perfect (p&lt;0.01; r = 1.00). On the other hand, semen cation concentrations were positively correlated but not + significant among themselves except the correlation between Na and K (p&lt;0.01; r = 0.37) which was significant. Relating the semen cation concentration with sperm morphological + characteristics, it was discovered that potassium ion (K ) measured was negatively and significantly correlated with all the morphological attributes (DMT &amp; DH, r= -0.36 @ p&lt;0.01; CBT &amp; ACR, r= -0.38 @ p&lt;0.05; MPD, r= -0.32 @ p&lt;0.05). It was concluded that + + DH is highly associated with DMT while Na concentration was an indicator for K level in the semen as shown in the result. Also, the higher the potassium ion in semen, the lower the level of sperm morphological abnormalities and vice versa in Yankasa rams.

English

English

Effect of heavy metals arsenic, cadmium, and lead on the semen variables of dromedary camels (Camelus dromedarius)

In this study, there was investigation of the effect of heavy metals on the fertility of dromedary camels. Fourteen camels at the Camel Research Center, King Faisal University, and 41 infertile dromedaries admitted to the Veterinary Teaching Hospital were used for semen evaluation during the breeding season. Seminal plasma and blood serum were collected from all males until analysis. Concentrations of three heavy metals [arsenic (As), cadmium (Cd), and lead (Pb)] were determined in the seminal plasma and serum using an atomic absorption spectrophotometer. The results indicate there are differences (P < 0.05 – P < 0.01) in pH, sperm motility, sperm concentration, and sperm abnormalities between the fertile and infertile male camels. In seminal plasma, there were marked differences (P < 0.01– P < 0.0001) between the control and infertile male camels in As, Cd, and Pb concentrations. In serum, there were differences (P < 0.01 – P < 0.001) between the fertile and infertile camels in serum As, Cd, and Pb concentrations. There was a positive correlation (P < 0.05; r = 0.77 and r = 0.94, respectively) between serum and seminal plasma concentrations of both As and Cd in the infertile dromedaries. In the control group, there was a positive correlation (P < 0.05; r = 0.70) between seminal plasma concentrations of Cd and percent sperm abnormalities. In conclusion, relatively greater seminal plasma and serum concentrations of As, Cd, and Pb are associated with lesser values for semen quality variables and infertility in dromedary camels.

Kinematic and head morphometric characterisation of spermatozoa from the Brown Caiman (Caiman crocodilus fuscus)

The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals but there has been no application of this technique for reptilian spermatozoa. The aim of this study was to analyse sperm kinematic and morphometric variables in Caiman crocodilus fuscus semen samples and to determine whether there were sperm subpopulations. Four ejaculates from four sexually mature captive caimans were used for this study. A CASA-Mot and CASA-Morph system was used with an image acquisition rate of 50 Hz for 2 s of capture. The ISAS®D4C20 counting chambers were used and spermatozoa incubated at 25 °C. Total and progressive motilities did not differ among animals (P > 0.05). There was a significant animal effect in the model with respect to sperm morphometry, and kinematic indices including linearity (LIN) and straightness (STR) (P < 0.05). Results for principal component (PC) analysis indicated variables were grouped into four components: PC1 related to velocity, PC2 to progressivity, PC3 to oscillation and PC4 to sperm path cross-linking. Subpopulation (SP) structure analysis indicated there were four groups, namely, rapid non-progressive (SP1), slow non-progressive (SP2), rapid progressive (SP3) and medium progressive (SP4), representing 14.5%, 45.4%, 18.7%, and 21.4% respectively. Findings in the present study indicate the importance of continuing development of reliable protocols regarding the standardisation of computer-based semen analyses in reptilian species.

Cryopreservation of Bangladeshi ram semen using different diluents and manual freezing techniques

A study was conducted to establish a sustainable and effective manual freezing technique for cryopreservation of Bangladeshi ram semen. Three diluents and freezing techniques were tested, both as treatment combinations (diluent × freezing technique) and fixed effects (diluent or freezing technique) on post-thaw sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI). Ten rams were selected, based on semen evaluation. Eight ejaculates were used for each treatment combination. Semen samples were diluted using a two-step protocol for home-made Tris-based egg yolk (20%, v/v) diluents: D1 (7% glycerol, v/v) and D2 (5% glycerol, v/v), and one-step for commercial diluent: D3 (Triladyl®, consists of bi-distilled water, glycerol, tris, citric acid, fructose, spectinomycin, lincomycin, tylosin and gentamycin) at 35 °C. Fraction-A (without glycerol) was added at 35 °C, and following cooling of sample to 5 °C (−0.30 °C/min), Fraction-B (with glycerol) was added. The diluted semen samples were aspirated into 0.25 ml French straws, sealed, and equilibrated at 5 °C for 2 h. The straws were frozen in liquid nitrogen (LN) vapour, in a Styrofoam box. The freezing techniques were; One-step (F1): at −15.26 °C/min from +5 °C to −140 °C; Two-step (F2): at −11.33 °C/min from +5 °C to −80 °C, and −30 °C/min from −80 °C-140 °C; and Three-step (F3): at −11.33 °C/min from +5 °C to −80 °C, at −26.66 °C/min from to −80 °C to −120 °C, and at −13.33 °C/min from −120 °C to −140 °C. Two semen straws from each batch were evaluated before and after freezing. The group F3D3 exhibited significantly higher (p < 0.05) post-thaw SM 63.1 ± 2.5%, SV 79.0 ± 2.1% and SPMI 72.9 ± 1.7%, whereas SAI 72.9 ± 1.7% was significantly higher (p < 0.05) in group F3D2. The freezing technique F2 and F3 had significantly higher (p < 0.05) post-thaw sperm values compared to F1. The post-thaw SM and SV were above 50% and 65% with the freezing technique F2 and F3 but differed non-significant. The SPMI 67.6 ± 2.0% and SAI 76.1 ± 1.4% were significantly higher (p < 0.05) with F3. Likewise, the diluent D2 and D3 had significantly higher (p < 0.05) post-thaw sperm values compared to D1. The post-thaw SM, SV and SPMI were above 50%, 65% and 55% with the diluents D2 and D3 but differed non-significant. The SAI 76.1 ± 1.1% was significantly higher (p < 0.05) with D3. We concluded that the use of a simple home-made Tris-based diluent containing 20% (v/v) egg yolk and 5% glycerol (v/v), two-step dilution and a three-step freezing technique is a sustainable and effective method for freezing ram semen. For further validation, the fertility of ewes artificially inseminated with the frozen semen will be observed.

Electrophoretic profile of seminal proteins and their correlation with in vitro sperm characters in Black Bengal buck semen.

Aim:This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability.Materials and Methods:Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson’s correlation coefficient, and two-way ANOVA was applied to find the individual buck differences.Results:Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze–thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen.Conclusion:Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.

Molecular markers of sperm quality

Light microscopic semen evaluation provides useful information about a given sperm sample, but due to its subjective nature has limited prognostic value for the reproductive performance of males or the outcome of assisted fertilization. Cryptic sperm abnormalities (occurring at the molecular level) are not easily detectable by light microscopy, but can be revealed by an array of biomarkers. The latter include fluorescent markers of acrosomal status, fluorochromes detecting altered sperm chromatin or DNA integrity, vital dyes revealing sperm mitochondrial activity, probes detecting apoptotic events, and antibodies detecting proteins that are either up- or down-regulated in defective spermatozoa. Many of the above biomarkers are best tested by flow cytometry, permitting rapid, automated, high throughput, objective measurement of the relative abundance of these biomarkers in semen. This review summarizes a strategy for the identification of novel male fertility/sperm quality biomarkers based on proteomic, biochemical and immunocytochemical analyses of defective spermatozoa. This approach identifies proteins or ligands uniquely associated with defective spermatozoa, regardless of whether they carry gross morphological defects or subtle, but critical hidden defects (e.g. DNA strand breaks) not detected with conventional, light microscopic analysis. Such markers, including ubiquitin, sperm thioredoxin SPTRX3/TXNDC8, 15LOX, and Lewis(y)-terminated N-glycans, are associated with poor semen quality and reduced fertility, warranting a designation of negative markers of fertility. The significance of sperm cytoplasmic droplet, a structure that accumulates several of the discussed biomarker proteins, is also discussed with regard to sperm quality and fertility.

Effects of Trehalose and Sucrose on Human Sperm Motility, Vitality and Morphology after Cryopreservation

Objective: To compare human sperm motility, vitality and morphology after cryopreservation among sucrose, and different trehalose concentrations.Material and Methods: A total number of 124 normozoospermic semen samples were collected. Each semen sample was divided into 4 portions, and cryopreserved in a human sperm-preserving medium along with cryoprotectants, including; 50 milimolar (mM) trehalose, 100 mM trehalose, 200 mM trehalose and 50 mM sucrose, respectively. All semen samples were frozen by using a vapor phase method. Post-thawed sperm motility, vitality and morphology were assessed. R program was used for data analysis. A p-value of &lt;0.05 was considered statistically significant.Results: Post-thawed semen evaluation indicated that 50 mM trehalose was better than 50 mM sucrose in all sperm parameters, which included progressive motility (p-value=0.037, total motility (p-value&lt;0.001), vitality (p-value&lt;0.001) and morphology (p-value&lt;0.001). The sperm parameters were not significantly different among 100 mM trehalose, 200 mM trehalose and 50 mM sucrose.Conclusion: The use of 50 mM trehalose, as non-permeating cryoprotectant, showed superior post-thaw sperm parameters over sucrose, and other trehalose concentrations.

Challenges associated with the development and transfer of assisted breeding technology in marsupials and monotremes: lessons from the koala, wombat and short-beaked echidna.

This reflective review describes how a research model, which was originally established for the successful AI of the koala (Phascolarctos cinereus), is currently being developed and extended to the wombat (Lasiorhinus latifrons and Vombatus ursinus) and short-beaked echidna (Tachyglossus aculeatus). The research model centres around the establishment of an AI program and involves: (1) semen collection, evaluation and preservation, requiring an understanding of male reproductive physiology and gamete biology; (2) timing of insemination, based on a knowledge of oestrous cycle and gestation physiology and oestrous behaviour; and (3) the appropriate placement of semen, which relies on an accurate description of female reproductive anatomy. Published and unpublished studies of assisted breeding technology (ABT) development in all three species of Australian mammals reported in this review (koala, wombat and short-beaked echidna) clearly demonstrate the importance of recognising species-specific variation in reproductive biology. Even in closely related species, such as the koala and wombat, subtle differences in reproductive physiology can hinder the transfer of ABT across species. Significant progress in marsupial and monotreme ABT will also require adequate access to captive wildlife colonies (zoos, university collections and private partners) in sufficient numbers in order to conduct quality science.

Cryopeservation Induced Alteration in Sperm Morphology in Hariana Bull Semen

Present study was conducted to evaluate the effect of semen cryopreservation on sperm morphology. For this 4 Hariana bulls were selected and 8 semen ejaculates from each bull were collected through artificial vagina method at twice a week interval. After preliminary semen evaluation, suitable semen samples were diluted with glycerolated egg yolk TRIS diluter. Semen samples were cryopreserved following the equilibration of 4h at 5˚C and finally stored into liquid nitrogen container. For sperm morphology head length, head width, base width, head area, mid piece length, tail length, total length, head ellipticity, head elongation and head regularity was measured in fresh as well as cryopreserved semen samples. Findings of the study revealed that there is a significant (P< 0.05) reduction in head area and other measurements after cryopreservation. The study was concluded with that cryopreservation induces various morphological changes in sperm which might be responsible for reduced fertility of frozen thawed spermatozoa.