Macro- and microelements in serum and seminal plasma as biomarkers for bull sperm cryotolerance

ABSTRACT Background Wide variation in fertility rates is observed when using frozen bull semen, even when the bulls have met quality standards for semen production. Therefore, a simple and reliable test to assess the freezing potential of bull semen based on the analysis of fresh semen or blood would be of great value. Attention is now turning to assessment of seminal plasma components such as proteins and elements. In the present study, the concentrations of macro- and microelements in fresh bull semen plasma and in serum and their correlation with quality characteristics of fresh semen and with semen quality after freezing and thawing were determined. Ejaculates were collected from 30 mature bulls, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability and DNA fragmentation were determined on the day of collection and after freezing and thawing. The concentrations of macroelements (Na, Mg, K and Ca) and microelements (Cu, Fe, Zn and Se) were determined in the seminal plasma and serum. The semen samples were classified into satisfactory and unsatisfactory groups according to the fresh semen quality. Results Zinc and Se levels measured in serum were associated with almost all fresh and frozen-thawed semen quality characteristics, while Fe levels were associated only with acrosomal defects in fresh semen. Zinc and Fe levels in fresh seminal plasma were associated with various quality characteristics of fresh and frozen-thawed semen, while Se level in fresh seminal plasma was not associated with any of the semen quality characteristics. Conclusions Microelements were shown to be useful as biomarkers involved in the analysis of bull sperm quality and could be used as an additional tool to predict bull semen quality after freezing and thawing. Our results confirm that the analysis of Zn and Se levels in serum and Zn, Cu and Fe levels in fresh seminal plasma can provide information to discriminate between bull semen samples with spermatozoa with high or low cryotolerance.

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Spectrophotometric Analysis of the Resazurin Reduction Test as a Tool for Assessing Canine Semen Quality

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2013-0049 ◽

2013 ◽

Vol 57 (2) ◽

pp. 281-285 ◽

Cited By ~ 1

Author(s):

Rafał Strzeżek ◽

Krystyna Filipowicz ◽

Marta Stańczak ◽

Władysław Kordan

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Semen Quality ◽

Membrane Integrity ◽

Spectrophotometric Analysis ◽

Normal Morphology ◽

Additional Tool ◽

Reduction Test ◽

Highly Correlated

Abstract The resazurin reduction test (RRT) was subjected to spectrophotometric analysis to evaluate the quality of canine semen. Twenty four samples of canine semen were analysed. The absorption peaks for resazurin and resorufin were determined at 615 and 580 nm, respectively. The RRT ratio (RRTsperm-the ratio for samples containing spermatozoa, RRTplasma-the ratio for samples containing seminal plasma) was calculated by dividing the absorbance at 580 nm by the absorbance at 615 nm. Spearman’s correlation test was used to determine the significance of correlations between the analysed sperm parameters and the results of the resazurin reduction assay. The RRT ratio was highly correlated with sperm motility (r=0.68, P<0.01), progressive sperm motility (r=0.61, P<0.01), the subpopulation of cells with rapid velocity (r=0.72, P<0.01), and the subpopulation of cells with medium velocity (r= -0.54, P<0.05). A negative correlation was observed between the reducing capacity of seminal plasma vs. sperm with plasma membrane integrity (r= -0.60, P<0.01) and sperm with normal morphology (r= -0.58, P<0.01). The RRT test can be used as an additional tool for evaluation of the quality of canine semen.

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113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab113 ◽

2006 ◽

Vol 18 (2) ◽

pp. 164

Author(s):

M. Thys ◽

A. Van Soom ◽

J. Dewulf ◽

T. Rijsselaere ◽

A. de Kruif

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Univariate Analysis ◽

Membrane Integrity ◽

Egg Yolk ◽

Freezing And Thawing ◽

Bovine Sperm ◽

Sperm Characteristics ◽

Group 2 ◽

Group 1

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

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Effect of inbreeding depression on bull sperm quality and field fertility

Reproduction Fertility and Development ◽

10.1071/rd15324 ◽

2017 ◽

Vol 29 (4) ◽

pp. 712 ◽

Cited By ~ 6

Author(s):

Jesús Dorado ◽

Rosa Morales Cid ◽

Antonio Molina ◽

Manuel Hidalgo ◽

Julia Ariza ◽

...

Keyword(s):

Inbreeding Depression ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Calving Interval ◽

Bull Semen ◽

Head Displacement ◽

Highly Active ◽

And Function

The present study investigated the effect of inbreeding depression on sperm quality using automated and objective methods and subsequent effects on beef bull field fertility. Individual inbreeding coefficient (F) values and field fertility data were determined using a dataset of AI bulls belonging to the Spanish Retinta Breeders Association (Asociación Nacional de Criadores de Ganado Vacuno Selecto de Raza Retinta (ANCRE)). Animals were clustered in two groups according to the F values as follows: (1) a high inbreeding group (HI; F ≥ 13.5%, mean 16.3); and (2) a non-inbreeding group (NI; F = 0%). In total, 17 different assessments were performed in both experimental groups, including evaluation of sperm morphology, acrosomal and DNA status, sperm plasma membrane integrity and function (hypo-osmotic swelling test), 10 kinetic parameters and the structure of sperm subpopulations. Sperm morphology, acrosomal and DNA status and osmotic tolerance were similar in both groups. Three velocity parameters (curvilinear velocity, straight line velocity and average path velocity) and the amplitude of lateral head displacement were higher in HI (P < 0.05). Cluster analysis of kinematic parameters revealed three different sperm subpopulations (sP1, sP2 and sP3), with the proportion of the sP1 population (highly active but non-progressive spermatozoa) being significantly (P < 0.05) higher in the HI group. Field fertility was assessed using two calving record datasets. In a smaller database including only bulls evaluated in the present study, there was a significant increase in the calving interval of cows sired with HI bulls. Conversely, in an extended genetic analysis of the ANCRE database, inbreeding only explained a small part of the variation in calving interval, and the results of regression analysis were not significant among bulls. The findings of the present study suggest that high inbreeding levels have a moderate effect on bull semen quality, with an increased percentage of highly active but non-progressive spermatozoa, but only when F values reached a certain threshold. This motility pattern could explain, in part, the higher calving interval produced by inbred bulls under field conditions.

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Novel insights into the role of cell-free seminal mRNAs on semen quality and cryotolerance of spermatozoa in bulls (Bos taurus)

Reproduction Fertility and Development ◽

10.1071/rd16290 ◽

2017 ◽

Vol 29 (12) ◽

pp. 2446 ◽

Cited By ~ 7

Author(s):

Munivenkatappa Shilpa ◽

Sellappan Selvaraju ◽

Venkataswamy GirishKumar ◽

Sivashanmugam Parthipan ◽

Krishnan B. Binsila ◽

...

Keyword(s):

Seminal Plasma ◽

Reproductive Performance ◽

Fas Ligand ◽

Bos Taurus ◽

Semen Quality ◽

Membrane Integrity ◽

Bone Morphogenetic Protein 2 ◽

Death Domain ◽

Bull Semen ◽

Morphogenetic Protein

The aim of the present study was to ascertain the effectiveness of seminal plasma mRNAs as markers to assess the reproductive performance of bulls. Semen samples (33 ejaculates) from 11 bulls were evaluated for sperm kinematic and functional parameters. Total RNA was isolated from cell-free seminal (cfs) using TRIzol LS reagent and the concentration of cfs-RNA was 24.4 ± 2.3 µg mL−1 seminal plasma. The cfs-RNA was fragmented to a size of 25–500 bp. Of the cfs-mRNAs screened using real time PCR, expression of protamine 1 (PRM1) was positively (P < 0.05) associated with the mitochondrial membrane potential of raw semen, whereas expression of Fas Ligand (FASLG) was negatively (P < 0.05) associated with sperm velocity, membrane integrity and chromatin distribution in post-thaw semen samples. The percentage of Type A spermatozoa (amplitude of lateral movement of head >2.5 μm and straightness >85%) in raw semen was positively (P < 0.05) associated with bone morphogenetic protein 2 (BMP2), ubiquitin conjugating enzyme E2D3 (UBE2D3), tumour-associated necrotic factor-associated death domain (TRADD) and caspase-3 (CASP3) expression. Nerve growth factor (NGF) expression was positively (P < 0.05) associated with the maintenance of post-thaw functional membrane integrity in spermatozoa and could be used to assess the cryotolerance of bull semen. In conclusion, the expression of cfs mRNAs can be used to assess the reproductive performance of males and to predict the sensitivity of spermatozoa to cryoinjury.

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PENGARUH METODE PENCUCIAN SPERMATOZOA SAPI ACEH TERHADAP MOTILITAS, PERSENTASE HIDUP, DAN INTEGRITAS MEMBRAN PLASMA UTUH SPERMATOZOA (The Infuence of Aceh Bull Spermatozoa Washing Method on Spermatozoa Motility and Plasma Membrane Integrity of Intact Spermatozoa)

Jurnal Medika Veterinaria ◽

10.21157/j.med.vet..v9i2.3808 ◽

2015 ◽

Vol 9 (2) ◽

Author(s):

Listin Handayani ◽

Dasrul Dasrul ◽

Muslim Akmal ◽

Cut Nila Thasmi ◽

Hamdan Hamdan ◽

...

Keyword(s):

Plasma Membrane ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Quality Parameters ◽

Control Group ◽

Plasma Membrane Integrity ◽

Spermatozoa Motility ◽

Sperm Washing ◽

Fresh Semen

This study aimed to determine the effect of sperm washing by swim up and centrifugation in isotonic medium on sperm quality of aceh bull. In this study, fresh semen from healthy male aceh bull aged 3-4 months was collected using artificial vagina. Immediately after semen collection, fresh semen quality was examined macroscopically and microscopically. Subsequently, sperm washing was performed by centrifugation and swim up in sperm washing medium. Group 1 (P0) as control group, cement washed with isotonic solution (andromed medium: saline solution) with ratio of 1:8. 2. Group 2 (P1), cement was separated by centrifugation method, group 3 (P2), all cement was separated by swim up method then examined the sperm quality sperm washing results. Each treatment was repeated 5 times. Quality parameters measured were the percentage of spermatozoa motility, sperm viability, and plasma membrane integrity intact spermatozoa. Data were analyzed with analysis of variance one-way pattern, followed by Duncan's multiple test. The results showed the mean ± SD percentage of sperm motility of each treatment group (P0; P1; P2) respectively amounted to 72.00±3.74, 66.40±4.77, and 73.60±3.29%. The percentage of viability was 72.00 ±3.74%, 66.40±2.88%, 71.80±2.17%. The percentage of plasma membrane integrity is intact spermatozoa was 68.20±1.79%, 57.20±3.77%, 69.00±2.00%. Results of this study showed that the percentage of motility, live spermatozoa and plasma membrane integrity intact after separation by swim-up method were significantly different (P <0.05) compared with no separation.Key words: spermatozoa quality, aceh bulls, centrifugation, swim up

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Quality of ram spermatozoa separated with modified swim up method

Bangladesh Veterinarian ◽

10.3329/bvet.v33i2.36459 ◽

2018 ◽

Vol 33 (2) ◽

pp. 62-70 ◽

Cited By ~ 2

Author(s):

A Hossain ◽

MM Islam ◽

F Naznin ◽

RN Ferdousi ◽

FY Bari ◽

...

Keyword(s):

Semen Quality ◽

Membrane Integrity ◽

Quality Parameters ◽

Fluid Medium ◽

Normal Morphology ◽

The Mean ◽

Mass Activity ◽

Fresh Semen ◽

Artificial Vagina

Semen was collected from four rams, using artificial vagina and viability%, motility% and plasma membrane integrity% were measured. Fresh ejaculates (n = 32) were separated by modified swim-up separation using modified human tubal fluid medium. Four fractions of supernatant were collected at 15-minute intervals. The mean volume, mass activity, concentration, motility%, viability%, normal morphology and membrane integrity% (HOST +ve) of fresh semen were 1.0 ± 0.14, 4.1 ± 0.1 × 109 spermatozoa/ml, 85.0 ± 1.3, 89.4 ± 1.0, 85.5 ± 0.7, 84.7 ± 0.5 respectively. There was no significant (P>0.05) difference in fresh semen quality parameters between rams. The motility%, viability% and HOST +ve % of first, second, third and fourth fractions were 53.4 ± 0.5, 68.2 ± 0.3, 74.8 ± 0.3 and 65.5 ± 0.4; 55.5 ± 0.4, 66.2 ± 0.4, 74.5 ± 0.3 and 73.6 ± 0.3 and 66.7 ± 0.5, 66.8 ± 0.5, 65.2 ± 0.4 and 74.7 ± 0.5 respectively. The motility%, viability% and membrane integrity% of separated semen samples differed significantly (P<0.05) between four fractions. The mean motility% and viability% were significantly higher (P<0.05) in third fraction (74.8 ± 0.3%), whereas the mean HOST +ve% was significantly higher (P<0.05) in fourth fraction (74.7 ± 0.5). All quality parameters of separated spermatozoa were significantly (P<0.05) lower than that of fresh semen. The pregnancy rates were higher with fresh semen (71%) in comparison to that of separated sample (57%).Bangl. vet. 2016. Vol. 33, No. 2, 62-70

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Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

Reproduction Fertility and Development ◽

10.1071/rd08238 ◽

2009 ◽

Vol 21 (4) ◽

pp. 571 ◽

Cited By ~ 23

Author(s):

T. Leahy ◽

J. I. Marti ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Seminal Plasma ◽

High Speed ◽

Sperm Quality ◽

Membrane Integrity ◽

Weight Fraction ◽

Protein Supplementation ◽

Freezing Process ◽

Spermatozoa Motility ◽

Functional Integrity

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

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The Effect of Glutathione on The Quality of Aceh Local Catfish (Clarias gariepinus) Spermatozoa After Cryopreservation

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v12i1.22353 ◽

2020 ◽

Vol 12 (1) ◽

pp. 125-131

Author(s):

Raudhah Mahfudhah ◽

Kartini Eriani ◽

Zainal Abidin Muchlisin ◽

Cut Ruhul Muthmainnah

Keyword(s):

Plasma Membrane ◽

Clarias Gariepinus ◽

Membrane Integrity ◽

Dna Integrity ◽

Freezing And Thawing ◽

Randomized Design ◽

Fetal Bovine ◽

Fresh Semen ◽

Different Doses

The cryopreservation process might reduce the quality of spermatozoa due to an increase in the production of reactive oxygen species (ROS) compounds during cooling, freezing, and thawing. The quality of spermatozoa can be maintained by adding glutathione as an exogenous antioxidant into cryo-diluent. This study aimed to examine the effect of the addition of different doses of glutathione in cryopreservation of Aceh Local catfish (Clarias gariepinus) spermatozoa after freezing. The method used was a completely randomized design (CRD) with four treatments and four replications. Fresh semen was diluted in Ringer, 15% DMSO, and 20% Fetal Bovine Serum (FBS) and then added with glutathione antioxidants of 0.0 mgL-1, 0.5 mgL-1, 1.0 mgL-1, and 2.0 mgL-1. The parameters observed in this study were motility, integrity of the plasma membrane, fertility, and DNA integrity. The results showed that the concentration of glutathione had no effect on motility, integrity ofthe plasma membrane, or fertility, but had an effect on DNA integrity. The highest percentage of motility and plasma membrane integrity respectively was 40.50% (P3) and 70.87% (P2). Furthermore, the assessment of DNA integrity showed that there was no DNA fragmentation both treatments and fresh spermatozoa. This research is the first study regarding glutathione supplementation in cryo-diluent of Aceh Local catfish spermatozoa. Finally, the results obtained can provide information about the exact concentration of glutathione in the extender on the quality of spermatozoa of Aceh Local catfish (C. gariepinus) after the cryopreservation process. These results can also increase the success of fertility be used by the seed hall unit and the aquaculture industry to increase the productivity and supply high quality seeds.

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Effect of Supplementation of Polyvinylpyrrolidone in Extender on Buffalo Semen Parameters

Pakistan Journal of Zoology ◽

10.17582/journal.pjz/20181230161216 ◽

2020 ◽

Vol 53 (1) ◽

Author(s):

Bushra Ismail Khan ◽

Shamim Akhter ◽

Sanwal Aslam ◽

Rabea Ejaz

Keyword(s):

Sperm Motility ◽

Semen Quality ◽

Membrane Integrity ◽

Bubalus Bubalis ◽

Semen Parameters ◽

Suction Pump ◽

Bull Semen ◽

Buffalo Semen ◽

Live Sperm ◽

And Control

The current study was planned to evaluate the supplementation of Polyvinylpyrrolidone in extender on cryopreservation of Nili-Ravi buffalo bull semen. The semen samples were collected from Nili-Ravi buffalo (Bubalus bubalis) bull kept at SPU Qadirabad, District Sahiwal, Pakistan. Qualifying semen ejaculates having motility >60%, volume >5-6ml and concentration >0.5 billion/ ml were diluted 50 × 106 motile sperm ml approximately at 37°C in Tris-citric acid extender supplemented with different concentrations of PVP (0.01, 0.05, 0.1mM). The extender without PVP was kept as control. Semen was stored at 4°C for a period of 2 h and kept at 4°C for 4h. Semen was filled in 0.5 ml French straws using suction pump at 4°C, plunged and stored in liquid nitrogen (-196°C). Semen straws were rewarmed at 37°C for 30 seconds and assessed for sperm motility, plasma membrane integrity (PMI), dead sperm percentage and the live sperm percentage. The data on the role of PVP on different parameters of semen quality were analyzed by using ANOVA and RCBD. Higher percentage (P< 0.05) of sperm motility (66.1±7.51 and 59.4±10.72) and PMI (72.9±5.39 and 75.7±6.5) was observed in extenders having 0.05 mM and 0.1mM PVP compared to extenders having 1.5mM PVP and control. The percentage acrosomal integrity was observed greater (P< 0.05) in extended semen containing 0.1mM (68.2±0.50) PVP compared to extenders having 0.01 and control.

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