Mid Piece Research Articles

COMPARATIVE STUDY OF FREEZING AND CHILLING EFFECTS ON SURVIVAL AND OXIDATIVE CHARACTERISTICS OF FUNAAB ALPHA (NORMAL FEATHER) CHICKEN SPERM

The impact of freezing and chilling on the semen of FUNAAB Alpha (Normal feather) chickens was examined in this study. Pooled semen collected from ten FUNAAB Alpha (Normal feather) chicken were diluted with Tris –based extender. Diluted semen was divided into two fraction, the first fraction was subjected to chilling at 4oC and the second fraction was subjected to freezing at 1oC for 24hrs. Thereafter semen were evaluated for viability, functional integrity and oxidative stress (Malondialdehyde concentrations MDA and seminal leukocytes). The result showed a consistent variation in the viability and functional integrity. However, Sperm motility and livability preserved at 4oC was significantly higher (p<0.05) than those preserved at 1oC. Abnormal sperm (head, mid piece and tail) recorded for those preserved at 4oC was significantly lower (p<0.05) than those preserved at 1oC. The findings indicate that sperm viability, functional integrities and oxidative stress parameters of FUNAAB alpha (Normal feather) were better preserved by chilling compared to freezing method.

The Cby3/ciBAR1 complex positions the annulus along the sperm flagellum during spermiogenesis.

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.

Semen characterization of Dang Surat Thai native chicken

Native Dang Surat chicken play an important role in poultry breeding of southern Thailand but their production performance falls short of commercial counterparts. Nevertheless, native birds have superior disease resistance while meat texture and taste of eggs are popular among Thais. Husbandry of native breeds are confined to smallholders who can only fulfil local niche outlets. To satisfy the wide-spread demand throughout Thailand and further afield, local-breed sires are crossed with high productivity hens. To maintain a constant supply of F1 hybrids, a colony of Dang Surat cocks are maintained by the Department of Livestock who select by phenotypes, not semen quality that determines male productivity. Accordingly, semen volume, pH, color and consistency, sperm motility, concentration, viability were measured. Morphology was also assessed by using eosin-nigrosin staining. Semen was slightly alkaline (pH, 8.0±0.1), mean volume=265±23 μL, had high sperm concentrations, 2740±76 x106/ml, of which 98.9±0.1% were viable. Abnormal sperm heads, tails and mid pieces, cytoplasmic droplets and detached heads comprised 1.5±0.3, 3.7±0.4, 0.3±0.1 and 0.8±0.1%, respectively. The findings showed normal seminal fluids and sperm parameters similar to commercial poultry semen in general. Our data will help develop efficient cockerel selection, natural flock breeding plan, and assisted reproduction by cryopreservation and artificial insemination to better meet market demands.

Detection of Chlamydia trachomatis inside spermatozoa using flow cytometry: Effects of antibiotic treatment (before and after) on sperm count parameters

There is increasing evidence that Chlamydia trachomatis (CT) infection can directly affect male fertility. However, only few have investigated the effects of CT on semen parameters, and mostly with inconclusive results. The main aims of this study were to identify CT inside spermatozoa, and the possible pre and post antibiotic treatment effects on the overall semen parameters. We developed a flow cytometric method for the detection of CT inside spermatozoa (SPI™). Briefly, sperm cells were fixed, membrane permeabilized and DNA was loosened using DNAse. Sperm cells were incubated with a primary monoclonal antibody against CT and with a secondary fluorescent antibody (vs primary), and analysed using a flow cytometer. Of 2415 infertile individuals, 48.61% were found positive for CT. 170 CT+ samples were included in the CT antibiotic treatment study. 78.82% (134/170) of the CT+ showed a significant reduction in the percentage of the iCT infected spermatozoa after the antibiotic treatment; 59.70% (80/134) decreased to non–detectable levels. Spermcount data were also recorded. Spermatozoa morphology (normal and teratozoospermia index, TZI) and motility (fast progressive and non–progressive spermatozoa) were statistically significant altered in CT+ pre–treatment vs control group. CT antibiotic treatment showed statistically significant effects on normal spermatozoa morphology, mid–piece and tail defects, and TZI. The study demonstrated that semen flow cytometric analysis of semen could be a valuable tool for faster and accurate identification of individuals with asymptomatic CT infection. It also identified a positive effect of antibiotic therapy on semen parameters, that could help males with infertility.

An investigation on cauda storage of sperm in hamster epididymis

Abstract Immature testicular sperm of mammals acquire the potential to move in a forward direction during their journey through excurrent ducts, efferent ductules and the epididymis. The present study aimed to determine the sperm storage time using the hamster cauda epididymis. Ligation of the left epididymal tubule at the junction between the distal corpus and the proximal cauda was carried out to determine the storage time. The right epididymis was left unaltered as the control. On days 3, 12, 15, 24, 28, 32, and 40 after ligation of the cauda, experiments were carried out and repeated five times. Sperm total count and mortality were determined using the haemocytometer counting method and sperm viability was assessed with the live/dead viability kit. The morphology of cauda sperm was observed by preparing sperm smears from each animal. Data were analyzed using SPSS and all values were expressed as mean ± SEM. On day 40, the total number of cauda sperms was reduced remarkably. In the experimental groups and in the control, 3–6% of sperm motility was maintained until day 40. By day 3, the percentage of live sperm was 50% and by the 40th day, it was decreased up to 10%. In the control group, the live sperm percentage was 24% by the 40th day (). By day 32, 76% of the cauda spermatozoa appeared abnormal with head defects, mid piece and neck defects and multiple defects. Findings of the present study indicate that cauda sperm storage time is more than 40 days. Motility, viability and morphology of these spermatozoa were decreased remarkably during this storage time.

EFFECT OF VITAMIN E SUPPLEMENTATION ON SPERM MORPHOLOGY AND SEMEN CHARACTERISTICS OF RABBIT BUCKS

Twenty mixed breed rabbit bucks aged between 6–8 weeks with an average weight of 861g were randomly assigned to four (4) dietary treatments with five (5) bucks per treatment in a Completely Randomized Design to determine the effect of feeding graded levels of Vitamin E on sperm morphology and semen characteristics of rabbit bucks. The rabbits received graded levels of vitamin E/kg feed (0mg/kg, 20 mg/kg, 40 mg/kg and 60 mg/kg for treatments 1, 2, 3 and 4, respectively). Feed and water were given to the rabbit bucks ad libitum during the experiment. The semen collected was taken to laboratory for sperm morphology and semen characteristics and the data collected were analysed using general linear model procedure of statistical analysis system (SAS). The results revealed that the mean number of normal cells, detached head and mid piece defect were not significantly (P>0.05) different across the treatments. However, the percent abnormal defects percent of free tail and coiled tail were significantly (P<0.05) lower for rabbits fed 20 and 60 mg/kg and the least bent tail was obtained in 60 mg/kg diet. The results for semen characteristics had significant (P<0.05) differences in all the parameters except for semen volume and pH. The results showed that those fed 60 mg/kg of vitamin E diet gave significantly (P<0.05) higher sperm motility, sperm concentration, live ratio and low dead ratio (87.50 %, 232x106, 87.50 % and 12.00 %) respectively. Therefore, supplementation of vitamin E at 60mg/kg in the diet of rabbit bucks is recommended

Detection of DNA Damage in Frozen Bovine Semen Using Eosin Staining.

<b>Background and Objective:</b> Early detection of DNA defects in spermatozoa is vital because genetic abnormalities are associated with infertility and miscarriage. Eosin is an acidic stain with negatively charged chromatolytic components also found in the cytoplasm of spermatozoa. This study aimed to observe the effectiveness of eosin staining in detecting spermatozoa DNA deterioration. <b>Materials and Methods:</b> A total of four-hundred frozen beef straws from Simmental, Limousin and Brahman cattle and Ongole crossbreeds, were obtained from the Singosari National Artificial Insemination Center, Malang, Indonesia.Hydrogen peroxide was used to degrade frozen sperm. In addition, the sperm was subjected to three treatments: Agarose trapping, membrane lysis and eosin staining. <b>Results:</b> Damaged bull spermatozoa stained with eosin were bright red in color around the head and mid piece, whereas the head of undamaged spermatozoa were darker red. The average value of damaged DNA was 89.08±3.27% as assessed with eosin staining. <b>Conclusion:</b> Eosin staining effectively detects DNA damage in bull spermatozoa, but extended effectiveness tests are considered necessary.

Morphometry and molecular characterisation of semen in four carp species

Present study aimed to compare the morphometric features of sperm and seminal protein profile of four cyprinid species viz. Labeo rohita (Hamilton, 1822), Labeo fimbriatus (Bloch, 1795), Cyprinus carpio (Linnaeus, 1758) and Puntius carnaticus (Jerdon, 1849) in order to elucidate quantitative characteristics of sperm morphology and seminal protein profiles within the family. Morphological studies revealed that carps have uniflagellate, acrosome less sperm with an ovoid head and irregular shaped mid piece. Size of sperm in terms of length and breadth showed significant variation among the four species studied. The sperm and seminal protein profiles of Puntius carnaticus differed from other three groups with the presence of proteins with molecular mass of 15 and 49 kDa and absence of four proteins equivalent to molecular mass of 13, 20, 21 and 23 kDa. Two dimensional gel electrophoresis (2D-GE) analysis of sperm protein profile between Puntius carnaticus and Labe orohita revealed presence of 237 protein spots. Keywords: Cyprinid, Morphometry, Protein profiling, SDS-PAGE, Sperm

Collapsed mitochondrial cristae in goat spermatozoa due to mercury result in lethality and compromised motility along with altered kinematic patterns

Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031–1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.

Incidence of sperm Abnormalities in Ejaculates of Breeder cocks fed Rations Containing Different Levels of Cottonseed Meal

A seven week study was conducted to determine the effect of three dietary levels of cottonseed meal (0%, 15%,30%) on the occurrence of spermatozoa morphological abnormalities in mature Rhode Island White (RIW) breeder cocks. Abnormalities of the acrosome, sperm head, cytoplasmic droplets and mid piece and sperm tail were investigated in 180 ejaculates collected between October and November. All types of abnormalities were variable between treatments and between extent of feeding (weeks). The values (2.90,5.30 and 8.63) on sperm head abnormalities were significantly different (P<0.01) between treatments and increased linearly with higher levels of cottonseed meal (CSM) in the diets. Abnormalities of the sperm tail were significantly variable (P<0.05) within treatment weeks with a linear increase from baseline value (2.00) lo about the 3r week post feeding (5.00). The mean total sperm morphological abnormalities in the treatment groups significantly increased linearly (P<0.01) as duration of feeding increased. It is suggested that breeder cocks should not be fed rations containing more than 15%CSM

Ultrastructural Sperm Flagellum Defects in a Patient With CCDC39 Compound Heterozygous Mutations and Primary Ciliary Dyskinesia/Situs Viscerum Inversus.

Introduction: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease characterized by structural or functional motile cilia abnormalities. Up to 40 different genes seem, at the moment, to be involved in the pathogenesis of PCD. A number of ultrastructural defects have also been reported in sperm flagella, but the sperm mitochondrial membrane potential (MMP) has never been described in these cases.Aim: The aim of this study was to report the sperm MMP and ultrastructural abnormalities of the sperm flagella found in a patient with PCD and situs inversus (Kartagener syndrome) and its characterization from the genetic point of view.Methods: Transmission electronic microscopy (TEM) analysis was used to evaluate flagella ultrastructure. The genetic testing was performed by next-generation sequencing. Sperm DNA fragmentation and MMP were also evaluated by flow cytometry.Results: We report here the case of an 18-year-old male patient with PCD and situs inversus and severe oligo-astheno-teratozoospermia. TEM analysis of his spermatozoa showed an abnormal connecting piece. The mid piece appeared abnormally thickened, with cytoplasmic residue, dysplasia of fibrous sheath, loss of the outer dynein arms (ODAs), truncated inner dynein arms, and supernumerary outer fibers. The percentage of spermatozoa with fragmented DNA was normal, whereas a high percentage of spermatozoa had low MMP, suggesting an altered mitochondrial function. The genetic analysis showed the presence of c.610-2A > G, p.Arg811Cys compound heterozygous mutations in the CCDC39 gene.Conclusion: The case herein reported suggests that the high percentage of sperm with low MMP may play a role in the pathogenesis of asthenozoospermia in patients with Kartagener syndrome. In addition, we report, for the first time, the missense variant p.Arg811Cys in the CCDC39 gene in a patient with Kartagener syndrome. Although in silico analysis predicts its damaging potential, its clinical meaning remains unclear.

A comparative study on seminal attributes of Sirohi and Beetal bucks

A total of 126 ejaculates, 21 from each of six bucks comprising three Beetal and three Sirohi bucks, collected by artificial vagina were used to study various semen characteristics. The semen characteristics viz., ejaculate volume, mass activity, initial sperm motility, live sperm, sperm concentration, sperm number per ejaculate, cold shock resistance index, live intact acrosome and sperm abnormalities were studied by conventional methods. The overall mean values of different characteristics of fresh semen in Beetal and Sirohi bucks for ejaculate volume, mass activity, initial sperm motility, live sperm, sperm concentration, sperm number per ejaculate, cold shock resistance index, live intact acrosome, head abnormalities, mid piece abnormalities, tail abnormalities and sperm with proximal and distal cytoplasmic droplet were 1.08 ± 0.05 and 1.18 ± 0.04 ml, 3.71+ and 3.92+, 82.94 ± 0.73 and 84.60 ± 0.61 per cent, 85.57 ± 0.52 and 86.03 ± 0.48 per cent, 3356.73 ± 83.06 and 3176.94 ± 53.92 million per ml, 3595.17 ± 122.08 and 3669.00 ± 126.42 million, 6.72 ± 0.22 and 6.77 ± 0.20 per cent, 85.18 ± 0.39 and 84.05 ± 0.41 per cent, 1.65 ± 0.13 and 1.50 ± 0.12 per cent, 0.25 ± 0.02 and 0.30 ± 0.03 per cent, 2.73 ± 0.18 and 2.53 ± 0.13 per cent, 0.43 ± 0.03 and 0.48 ± 0.04 per cent and 0.86 ± 0.12 and 0.61 ± 0.04 per cent respectively. The different semen characteristics did not differ significantly between breeds; however, the differences between bucks within the breed were significant for ejaculate volume, live intact acrosome, mid piece abnormalities, tail abnormalities and sperm with distal cytoplasmic droplet.

Specialized PIEZO-ICSI for low quality oocytes

In 1995, Kimura and Yanagimachi reported the usefulness of ICSI using Piezo-micro manipulator by applying a Piezo pulse which produced ultra-fast sub-micron forward momentum using uniquely shaped flat-tipped micropipettes with no bevel or spike (Piezo-ICSI). Hiraoka et al reported that Piezo-ICSI has advantage of high fertilization rate, low damage rate of oocyte at ICSI and high clinical outcome in 2015. However, we have worried about one problem in Piezo-ICSI, that is the volume of injected medium at ICSI. So, we developed a newly specialized Piezo-ICSI with sperm with a shortened sperm tail after cutting the tails to lessen the damage to the cytoplasm of these low-quality oocytes. We then investigated the effect of the specialized Piezo-ICSI. Prospective study to improve clinical outcome of Specialized Piezo-ICSI for Low quality oocytes. The sperm tail was cut with injection pipette a little below the mid piece then aspirate it injection pipette head first. The zona pellucida was penetrated using a weak piezo pulse (speed 1.5, intensity 1) and the tip of injection pipette was introduced forward to stretch the cytoplasmic membrane. A weaker pulse (speed 1.0, intensity 1) was added to break it and the sperm injected simultaneously. Pushing the sperm forward and aspiration of the medium injected at ICSI were unnecessary. Oocytes that received ICSI, Oocytes that survived after ICSI (%), Oocytes fertilized (%), ood quality day-3 embryos (%), Blastocysts (%), Clinical pregnancies (%) between conventional Piezo-ICSI and Specialized Piezo-ICSI were [512, 124] [435 (85),112 (90)] [409 (80),103 (83)] [266 (52),69 (56)] [230 (45),60 (48)] [27,31] respectively.

Oviductal extracellular vesicles interact with the spermatozoon\u2019s head and mid-piece and improves its motility and fertilizing ability in the domestic cat

Fertilization and early embryo development are regulated by a unique maternal-gamete/embryo cross-talk within the oviduct. Recent studies have shown that extracellular vesicles (EVs) within the oviduct play important roles in mediating this developmental process. Here, we examined the influence of oviductal EVs on sperm function in the domestic cat. We demonstrated that (1) EVs are enriched in proteins related to energy metabolism, membrane modification, and reproductive function; (2) EVs bound and fused with the membranes of the acrosome and mid piece; and (3) incubating sperm with EVs improved motility, fertilizing capacity of cat spermatozoa and prevented acrosomal exocytosis in vitro. These findings indicated that oviductal EVs mediate sperm function and fertilization in the cat and provides new insights to improve sperm cryopreservation and in vitro fertilization in the domestic and wild felids and human.

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.

Daily administration of a GnRH analogue enhances sperm quality in bucks during the non-breeding season

The aim of this study was to determine if daily administration of a GnRH analogue (buserelin acetate) to bucks during the non-breeding season increases testosterone concentration and improves sperm quality. Five bucks received a daily dose of buserelin for 10 days, starting on Day 0 (first administration), and another five bucks remained as controls. Testosterone concentrations were greater in treated than in control bucks during the first hours after buserelin administration (P = 0.05), but greater in controls 10 h later (P < 0.01). Sperm mass motility and percentage of motile sperm were greater in treated (3.9 ± 0.6 and 70.1 ± 7.9%, respectively) than in control bucks (1.0 ± 0.6, P < 0.01; 45.0 ± 7.9%, P < 0.05 respectively) on Day 4. Percentage of sperm with normal morphology tended to be greater in treated than in control bucks (81.8 ± 6.2% compared with 63.5 ± 6.4% respectively, P = 0.08). The treatment decreased the percentage of sperm with mid piece defect and with bent tail (7.0 ± 1.5% compared with 12.0 ± 1.5%; 8.0 ± 1.7% compared with 13.5 ± 1.7%, treated and control bucks, respectively, P = 0.05 for both). The square root percentage of sperm with loose but heads with normal structures tended to be less in treated than control bucks (1.3 ± 0.3% compared with 0.4 ± 0.3% respectively, P = 0.06). It was concluded that daily administration of buserelin during the non-breeding season led to a rapid increase in testosterone concentration and improved sperm quality.

Egg yolk enriched with polyunsaturated fatty acids (PUFAs) improves the shelf life of ram semen in liquid storage

The aim of the current study was to assess the ability of semen extenders using egg yolk enriched with different sources of PUFAs to increase the shelf life of the ram semen in liquid storage. Four sources of PUFAs: conjugated linoleic acid (CLA), olive oil, flaxseed oil and fish oil were included in a basal diet (50 g/kg) and were fed to thirty laying hens. Semen samples were collected twice a week from six sexually mature Afshari rams. Semen samples were pooled and extended with five Tris-based extenders containing egg yolk from a normal diet, or egg yolk from the four enriched diets. Diluted semen samples were cooled to 4 °C and stored for 120 h (h). Sperm parameters were assessed at 0, 96 and 120 h of liquid storage. Proportions of motile and viable sperm were highest in flaxseed oil and fish oil extenders at 0 (P < 0.05), 96 and 120 h post storage (P < 0.01) whereas sperm plasma membrane functionality was highest in flaxseed oil and fish oil extenders compared to that of the other extenders for all times (P < 0.05). Percent of spermatozoa bearing head abnormalities were lower (P < 0.05) in omega-3 based extenders (flaxseed oil, fish oil and CLA), while the mid piece and tail abnormalities were lower (P < 0.01) in flaxseed oil, fish oil and olive oil extenders. In conclusion, enrichment of egg yolk with polyunsaturated fatty acids can improve the shelf life of ram semen during liquid storage.

Cadmium effects on sperm morphology and semenogelin with relates to increased ROS in infertile smokers: An in vitro and in silico approach

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility.

Comparison of the external physical damages between laser-assisted and mechanical immobilized human sperm using scanning electronic microscopy

We aim to visualize the external physical damages and distinct external phenotypic effects between mechanical and laser-assisted immobilized human spermatozoa using scanning electronic microscopy (SEM). Human spermatozoa were immobilized mechanically or with laser assistance for SEM examination and the membrane integrities were checked on both types of immobilized spermatozoa. We found evidence of external damages at SEM level on mechanically kinked sperm, but not on laser-assisted immobilized sperm. Although no external damage was found on laser-assist immobilized sperm, there were two distinct types of morphological changes when spermatozoa were stricken by infra-red laser. Coiled tails were immediately formed when Laser pulse was applied to the sperm end piece area, whereas laser applied to the sperm principal piece area resulted in a sharp bend of sperm tails. Sperm immobilized by laser did not exhibit any morphological change if the laser did not hit within the on-screen central target zone or if the laser hit the sperm mid piece or head. Our modified membrane integrity assay revealed that the external membrane of more than half of the laser-assisted immobilized sperm remained intact. In conclusion, mechanical immobilization produced membrane damages whilst laser-assisted immobilization did not result in any external membrane damages besides morphological changes at SEM level.

Effect of foot and mouth disease vaccination on seminal traits in pure Holstein Friesian bulls

The present investigation was carried out to study the effect of foot and mouth disease vaccination on seminal traits of 30 Holstein Friesian (HF) bulls, 3-5 years old, maintained at BAIF Central Research Station, Uruli Kanchan, Pune, India. The study period was from August to September 2013 and semen quality traits were evaluated on each bull maintained under identical feeding and management regimes. A total of 180 semen ejaculates were collected with one ejaculate from each bull at 3 days interval in a week at 15 days before, 15 and 30 days after FMD vaccination. Raksha Ovac vaccine of Indian Immunologicals Ltd. was used for vaccination purpose. Seminal traits like fresh and post thaw sperm motility, sperm concentration, semen volume, live and dead count, plasma membrane integrity were evaluated for all the ejaculates. Data on seminal traits were analysed by using one way ANOVA. Results revealed that vaccination had significant (P less than 0.05) effect on post thaw motility and highly significant (P less than 0.01) deleterious effect on HOS test, head and mid piece abnormalities; however no effect was noted on ejaculate volume, sperm concentration, initial motility, viability and tail abnormalities. The observations showed that vaccination alters the seminal traits of pure HF bulls.