Initiation Of Sperm Motility Research Articles

Bovine Sperm Motility as Affected by Alpha Tocopherol and Ascorbic Acid during Storage

Our purpose was to determine the impact of ascorbic acid and α-tocopherol on bovine sperm motility following short-term storage or cryopreservation. Semen was collected from mature Angus bulls (n = 4). The experimental design was a randomized complete block, with bull serving as block, and treatments were structured as a 4 × 4 factorial with four concentrations of ascorbic acid (0, 5, 10, 20 mM) and four concentrations of α-tocopherol (0, 0.05, 0.5, 5 mM). Sperm motility characteristics were evaluated using computer assisted sperm analyses at 0, 4, and 8 h of incubation (39˚C) and post-cryopreservation. Initial sperm motility was (79.6% ± 1.6) and decreased to (6.1% ± 1.6%) after cryopreservation. Cryopreserved spermatozoa had lower (P 0.1) by α-tocopherol. Addition of 20 mM ascorbic acid to storage media decreased (P < 0.05) sperm velocity traits, but the addition of 5 or 10 mM ascorbic acid did not alter sperm velocity. Sperm cell oxidation following cryopreservation/post-thaw was affected by an interaction (P < 0.05) between concentrations of ascorbic acid and α-tocopherol. Stepwise regression models predicted (P < 0.05) post-thaw motility and velocity characteristics of cryopreserved spermatozoa. Our results suggest that adding ascorbic acid and α-tocopherol was not beneficial for short-term storage of spermatozoa; however, our results were inconclusive with regards to inclusion of ascorbic acid and α-tocopherol in egg yolk-based cryopreservation media.

Ergot Alkaloid Effects on Bovine Sperm Motility &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt;

Cattle in some parts of the world graze pastures that consist of tall fescue that may contain ergot alkaloid contamination. Those ergot alkaloids are associated with reduced reproductive rates in cattle. Our objective was to determine if ergot alkaloids [dihydroergotamine (DHET), ergonovine (EN), and ergotamine (ET)] directly affect bovine sperm characteristics. Spermatozoa were collected from mature Angus (n = 2) and Balancer (n = 4) bulls. Within bull, treatments were structured as a 3 × 5 factorial with three alkaloids (DHET, EN, and ET) and five concentrations of each alkaloid (0, 33, 66, 100, or 200 μM). Spermatozoa (25 × 106) were incubated (39˚C) in 1 mL of modified sperm medium. Sperm motility characteristics were evaluated using CASA (Hamiliton Thorne IVOS, Beverly, MA) at 0, 3, and 6 h after initial alkaloid exposure. Initial sperm motility was (69% ± 1.1%) and declined (P = 0.01) to (35% ± 2.6%) at 6 h. Sperm motility decreased (P < 0.05) with increasing concentrations of DHET and ET, but not EN. As concentration of ET or DHET increased all CASA sperm characteristics were altered. The interaction of alkaloid concentration and incubation length affected sperm velocity and head size; exposure to 200 μM of ET or DHET for six hours decreased (P < 0.05) both characteristics. Our results demonstrate that ergot alkaloids (ET and DHET) can directly alter bovine sperm motility and morphology, which adds to our understanding of how ergot alkaloids may hinder cattle reproductive rates.

Expression study of fertility related genes with respect to sperm motility, HOST and acrosome integrity in bovine semen

The present study was conducted for molecular evaluation of bull semen and to study its relationship with different semen characteristics. Fresh semen samples were collected from six breeding bulls. A total of six ejaculates were collected from each of six healthy breeding Jersey bulls at 4 days interval. Immediately after collection, the samples were subjected to physio-morphological and biomolecular evaluation. Percentage of Hypo-osmotic Swelling Test (HOST) positive spermatozoa and acrosome-intact sperms increased with an increase in initial sperm motility. Expression of Chaperonin Containing T-Complex protein 1, subunit 8 (CCT8) gene was found to be negatively correlated with sperm motility, whereas the expression of Adenylate Kinase 1 (AK1) gene did not show any significant relationship with sperm motility.

Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands.

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin-like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin-like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate Na -benzoyl-L-arginine-ethyl-ester-hydrochloride (BAEE), trypsin-like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) -agarose affinity column was used to independently identify trypsin-like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS-PAGE gels. Taken together, these data demonstrate the presence of trypsin-like activity and several trypsin-like proteins in the Culex male accessory glands.

Changes in external osmolality and ionic composition affect Megaleporinus obtusidens sperm motility

Understanding the effects of environmental factors on sperm motility characteristics can increase artificial reproduction efficiency in species that do not spawn naturally in captivity, such as Megaleporinus obtusidens. This study evaluated the effects of the osmolality (25, 85, 145, 205, 265, and 325 mOsm kg−1) and composition of activating solutions (NaCl, KCl, or fructose) on the percentage of motile sperm, and the swimming velocity and path straightness of M. obtusidens spermatozoa. The concentrations of major ions in the seminal fluid were also assessed and Na+ (74.46 mmol L−1), K+ (37.24 mmol L−1), and Cl− (114.29 mmol L−1) were the most abundant. When the activating solution was hypertonic (325 mOsm kg−1) compared to the seminal fluid (293 mOsm kg−1), sperm motility was completely inhibited. A wide range of osmolalities that initiated sperm motility were identified for all three solutions. Both, the percentage of motile sperm and the motility duration were reduced (P < .05) at extreme osmolalities. At 145 mOsm kg−1, the percentage of motile sperm remained high (>50%) up to 40 s after activation and the motile phase lasted for >50 s, regardless of the activating solution composition. Over the postactivation time, the curvilinear velocity and straightness were similar (P > .05) for fructose and NaCl solutions, whereas KCl solutions induced a higher (P < .05) curvilinear velocity, lower (P < .05) straight-line velocity, and a circular swimming motion in spermatozoa. Our results suggest that a reduction in osmolality, using both non-electrolyte and electrolyte solutions, is the main trigger for the onset of spermatic movement in M. obtusidens sperm.

Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3β, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3β, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3β are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3β does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

The effect of freezing on cryosurvival of yak sperm

A study was carried out to study the effect of freezing on cryosurvival of yak semen. Artificial insemination in yak is still in infancy. Semen cryopreservation and use of artificial insemination can be applied in yak husbandry for conservation and rapid multiplication of superior germplasm. Semen was collected from four adult yak bulls using artificial vagina method managed under uniform conditions. A total of 40 ejaculates comprising of 10 ejaculates each bull were collected following twice a week schedule and evaluated for fresh semen characteristics. The fresh yak semen characteristics viz. ejaculate volume (ml), mass activity (0-4), initial sperm motility (%), sperm concentration (x 106/ml), live sperm (%), sperm abnormality (%) and intact acrosome (%) were 3.10 ± 0.18, 3.53 ± 0.96, 83.89 ± 2.87, 1180.22 ± 42.32, 77.63 ± 4.23, 8.45 ± 3.33 and 93.61 ± 3.78 respectively. The ejaculates were diluted (1:10) with Tris extender consisting of 6.4 ml glycerol and 20 ml of fresh egg yolk. Straws were equilibrated at 5°C for 4 hours followed by exposure to liquid nitrogen vapour for 10 minutes and finally transferred to liquid nitrogen container for storage. The cryosurvival rate was studied after 7 days of storage in liquid nitrogen. The frozen semen was thawed in warm water (37°C) for 30 seconds for evaluation. Mean values of postthaw sperm motility (%), live sperm (%) and intact acrosome (%) in yaks were 55.67 ± 4.67, 65.62 ± 3.23 and 89.26 ± 3.67 respectively. In conclusion, yak semen has a better cryosurvival while freezing in tris extender with 6.4 per cent glycerol and 20 per cent egg yolk following an equilibration period of 4h.SAARC J. Agri., 15(2): 215-218 (2017)

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus)

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.

Protein phosphorylation in spermatozoa motility of Acipenser ruthenus and Cyprinus carpio

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Characteristics and freezability of Assam Hill goat semen

Assam Hill goat (AHG) is an important goat germplasm found in Assam and its adjoining areas of India. The study was designed with an objective to study the semen characteristics and freezability of AHG buck semen using Tris -Egg yolk-Citrate-Fructose diluent. The mean values of fresh semen characteristics in AHG bucks viz., ejaculate volume (ml), initial sperm motility (%), sperm concentration (x106/ml), live sperm (%), sperm abnormality (%), HOST-reacted sperm (%) and intact acrosome (%) recorded were 0.39 ± 0.01, 77.97 ± 0.73, 3201.00 ± 143.78, 83.02 ± 0.65, 7.66 ± 0.73, 66.95 ± 0.74 and 93.34 ± 0.51, respectively. Mean values for post-thaw semen characteristics i.e., sperm motility (%), live sperm (%), HOST-reacted sperm (%) and intact acrosome (%) were 55.39 ± 0.97, 71.01 ± 0.78, 54.77 ± 0.55 and 82.16 ± 0.43, respectively. It can be concluded that AHG bucks donate acceptable quality of semen which can be frozen successfully in Tris-Egg yolk-Citrate-Fructose diluents for using in Artificial Insemination.

Success in the acquisition of Bombyx mori sperm motility is influenced by the extracellular production of nitric oxide (NO) in the presence of seminal fluid nitric oxide synthase (NOS)

A trypsin-like protease called initiatorin is known to initiate sperm motility in the silkworm, Bombyx mori, but little is known about the signaling events leading to sperm flagellar beating. The aim of this study was to investigate whether this mechanism of sperm motility activation involves the signaling transmitter nitric oxide (NO). NO is produced from the amino acid L-arginine by the enzyme action of nitric oxide synthase (NOS; EC 1.14.13.39). Simple treatment of quiescent sperm with an NO donor (SNAP or NOC7) in vitro did not lead to activation of motility. Nevertheless, initiatorin- or trypsin-induced motility was blocked by pretreatment of sperm with either the NOS inhibitor L-NAME or NO scavenger carboxy-PTIO. These observations suggested that NO may play important physiological roles in the acquisition of sperm motility under the in vitro condition used here. Then, we investigated whether NO synthesis would occur in the spermatophore, a capsule containing spermatozoa that is created by the contents of various male reproductive glands and is the site of sperm maturation. The amounts of NO2− and NO3−, stable metabolites of NO, reached maximum values after enclosure in the spermatophore, a time when apyrene spermatozoa acquire vigorous motility. Moreover, RT-PCR and Western blotting analyses of NOS indicated that it is abundantly expressed in glandula (g.) lacteola of the virgin male ejaculatory duct, from which it is secreted to the seminal fluid and transferred to the female during mating. Previous studies demonstrated that free L-arginine is supplied de novo by a specific proteolytic reaction in which initiatorin participates during spermatophore formation (Osanai et al., 1987c). Based on these results, it can be presumed that the mixing of seminal fluid contents from each male reproductive organ during ejaculation induced NO production outside of the spermatid, and exogenous NO stimulated a signaling pathway involved in the activation of silkworm apyrene sperm.

Protein phosphorylation and ions effects on salmonid sperm motility activation

AbstractSperm motility is considered as a key factor allowing determination of semen quality and predicts fertilizing capacity. In many fish species, the spermatozoa are immotile in the testes and seminal plasma, and motility is induced when they are released in the aqueous environment. Initiation and activation of sperm motility are prerequisite processes for the contact and fusion of male and female gametes at fertilization. Many proteins are involved in the activation of sperm motility in many species. Cell signalling for the initiation of sperm motility in the salmonid fish has drawn much attention during the last two decades. In some species, protein phosphorylation process was shown to be involved in flagellar motility regulation. Hyperpolarization of the sperm membrane induces synthesis of cAMP (cyclic AMP), which triggers further cell signalling processes, such as cAMP‐dependent protein phosphorylation that finally initiates sperm motility in salmonid fish. Ions such as Na+, K+ and Ca2+ play also an important role in the activation of sperm motility in many species, more specifically in salmonids. Salmonid fish sperm motility can be suppressed by millimolar concentrations of extracellular K+, and dilution of K+ upon spawning is enough to trigger the cAMP‐dependent signalling cascade leading to motility initiation. This review aims to update the present knowledge about the roles of ions and protein phosphorylation process in the sperm motility activation in salmonids.

Sperm motility activation in the critically endangered booroolong frog: the effect of medium osmolality and phosphodiesterase inhibitors.

Effective activation of sperm motility is fundamental to successful artificial fertilisation; however, studies investigating optimal procedures in amphibians are lacking. This study found the optimal osmolality of activation media for sperm motility activation and evaluated the effect of phosphodiesterase (PDE) inhibitors on sperm activation and longevity in the critically endangered booroolong frog, Litoria booroolongensis. To assess the effect of medium osmolality (10, 25, 50, 75, 100 and 200mOsmolkg-1) and PDE inhibitors (control, 2.5mM caffeine, 5mM caffeine, 2.5mM pentoxifylline, 5mM pentoxifylline, 2.5mM theophylline and 5mM theophylline) on initial activation, percentage sperm motility and sperm velocity were quantified using computer-assisted sperm analysis. To assess the effect of PDE inhibitors (control, 2.5mM caffeine and 2.5mM theophylline) on sperm longevity, percentage motility and velocity were assessed hourly until 10h after activation. High (>60%) percentage motility was achieved in a broad range of activation-medium osmolalities (10-75mOsmolkg-1). PDE inhibitors did not have an effect on initial sperm motility or velocity, but caffeine and theophylline improved sperm longevity, significantly increasing motility and velocity at 8, 9 and 10h after activation. Data also show that sperm longevity in L. booroolongensis is extreme, with spermatozoa remaining motile more than twice as long as those of any other anuran amphibian.

Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (P<0.05). In addition, initial sperm motility was significantly higher in individuals with the GG or GA genotype than in individuals with the AA genotype (P<0.05). The findings of the present study suggest that the difference in semen quality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

Queen reproductive tract secretions enhance sperm motility in ants.

Queens of Acromyrmex leaf-cutting ants store sperm of multiple males after a single mating flight, and never remate even though they may live for decades and lay tens of thousands of eggs. Sperm of different males are initially transferred to the bursa copulatrix and compete for access to the long-term storage organ of queens, but the factors determining storage success or failure have never been studied. We used in vitro experiments to show that reproductive tract secretions of Acromyrmex echinatior queens increase sperm swimming performance by at least 50% without discriminating between sperm of brothers and unrelated males. Indiscriminate female-induced sperm chemokinesis makes the likelihood of storage directly dependent on initial sperm viability and thus provides a simple mechanism to secure maximal possible reproductive success of queens, provided that initial sperm motility is an accurate predictor of viability during later egg fertilization.

Role of potassium and pH on the initiation of sperm motility in the European eel

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pHi in the quiescent stage was also induced. Intracellular pH (pHi) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pHi (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

STUDY ON BODY WEIGHT AND SCROTAL CIRCUMFERENCE AND ITS CORRELATIONS WITH SEMEN PRODUCTION TRAITS IN CROSSBRED JERSEY BULLS

The present study was aimed to investigate the effects of body weight and scrotal circumference on semen production traits of 136 crossbred Jersey bulls, which were maintained at three frozen semen stations of Tamil Nadu, India. Semen production traits such as semen volume, sperm concentration, mass activity, initial sperm motility, post-thaw motility and number of doses per ejaculate were taken for this study. The overall mean values for semen volume (ml), sperm concentration (millions per ml), mass activity (0 to 5 scale), initial sperm motility (per cent), postthaw motility (per cent) and number of doses per ejaculate were 4.18 ± 0.02, 1052.80 ± 7.26, 2.09 ± 0.07, 63.36 ± 0.00, 50.52 ± 0.00 and 215.50 ± 2.01 respectively. From this study, it revealed that the body weight and scrotal circumference of bulls significantly influenced all semen production traits except mass activity. Bulls having a body weight between 350 to 550 kg produced better quality semen and more number of frozen semen doses per ejaculate. When the scrotal circumference was &gt;36 cm, majority of the semen production traits showed better values. The body weight was positively and significantly correlated with scrotal circumference, Semen volume and number of frozen semen doses produced per ejaculate. The scrotal circumference was positively and significantly correlated with semen volume, mass activity and number of doses per ejaculate; negatively and significantly correlated with initial sperm motiltiy.

Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant

Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.