Membrane Acrosome Integrity Research Articles

Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?

BackgroundIn humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs.MethodsForty Nellore bulls were divided into three age groups: 1.8 to 2 years – young bulls; 3.5 to 7 years – adult bulls; and 8 to 14.3 years – aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA).ResultsWe report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05).ConclusionThese results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

Effect of seminal plasma and butylated hydroxytoluene (BHT) concentration on ram sperm freezability

The effect of removing seminal plasma (SP) by centrifugation before ram semen cryopreservation was assessed. Simultaneously, we studied the supplementation of different concentrations of BHT as antioxidant (0.6mM, 2mM and 5mM) on the extender in order to find an optimal concentration in enhancing a successful ram sperm cryopreservation. Therefore, fresh ejaculates from 8 rams of approximately 2 years old were collected, immediately mixed in equal quantities and split into 2 aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600×g for 10min, while the other aliquot was kept unwashed. Afterwards, washed and unwashed sperm were re-suspended in one of different TCG-based media with 5% glycerol and 15% powdered egg yolk (PEY) supplemented or not with BHT and/or SP according to the treatments: 1) washed sperm; 2) washed sperm+0.6mM BHT; 3) washed sperm+2mM BHT; 4) washed sperm+5mM BHT; 5) washed sperm+0.5mL of SP; 6) washed sperm+5mM BHT+0.5mL of SP; 7) unwashed sperm and 8) unwashed sperm+5mM BHT. Afterwards, sperm samples were refrigerated at 5° C for 4h before being packed in 0.25mL straws and frozen in nitrogen liquid vapors. After thawing, plasma and acrosomal membrane integrity and mitochondrial activity were determined by flow cytometry while sperm motility and morphometry by CASA (mean±SEM, n=6). Our results showed that seminal plasma removal enhanced sperm cryosurvival and the addition of BHT (5.0mM) improved most of the quality parameters of thawed washed and unwashed sperm, irrespective of the presence of SP in the extenders.

Effect of selenium supplementation on semen characteristics of Brazil's ram.

The aim of this study was to investigate the effects of different concentrations of oral supplementation with selenium (Se) upon ram sperm parameters. Thirty rams managed in stall under intensive system were used and divided into five groups (six animals per group) as follows: control group (G1) mineral mixture supplementation without Se, group 2 (G2) mineral mixture supplemented with 5mg/kg Se, group 3 (G3) supplemented with 10mg/kg Se, group 4 (G4) supplemented with 15mg/kg Se and group 5 (G5) supplemented with 20mg/kg Se. For each group, there was an adjustment period of 14days. The experimental period was 350days. Every 56days, the animals were weighed and semen samples were collected by electroejaculation. Semen analysis included volume, mass moviment, total motility, vigour, concentration and morphology. For plasmatic and acrosomal membrane integrity evaluation and mitochondrial membrane potential were used a combination of fluorescent probes. Differences between means values obtained by analysis of variance were verified by Tukey test with 5% probability. There was no statistical difference between treatment groups in relation to volume, mass moviment, total motility, vigour, concentration, plasma and acrosomal membrane integrity (p>.05). Sperm morphology was different between treatment groups, the G1 (0mg of selenium) had the highest percentage of major defects (11.11±1.11a ; p<.05). It was concluded that selenium decrease the percentage of sperm defects and did not directly influence on ram sperm volume, mass moviment, total motility, vigour, concentration and membrane integrity.

REAC technology as optimizer of stallion spermatozoa liquid storage

BackgroundREAC technology (acronym for Radio Electric Asymmetric Conveyor) is a technology platform for neuro and bio modulation. It has already proven to optimize the ions fluxes at the molecular level and the molecular mechanisms driving cellular asymmetry and polarization.MethodsThis study was designed to verify whether this technology could extend spermatozoa life-span during liquid storage, while preserving their functions, DNA integrity and oxidative status. At 0, 24, 48, and 72 h. of storage at 4 °C, a battery of analyses was performed to assess spermatozoa viability, motility parameters, acrosome status, and DNA integrity during REAC treatment. Spermatozoa oxidative status was assessed by determining lipid peroxidation, the activity of superoxide dismutase (SOD), and the total antioxidant capacity.ResultsDuring liquid storage REAC treated spermatozoa, while not showing an increased viability nor motility compared to untreated ones, had a higher acrosome (p > 0.001) and DNA integrity (p > 0.01). Moreover, the analysis of the oxidative status indicated that the mean activity of the intracellular superoxide dismutase (SOD) was significantly higher in REAC treated spermatozoa compared to untreated controls (p < 0.05), while the intracellular concentration of malondialdehyde (MDA), an end product of lipid peroxidation, at the end of the REAC treatment was higher in untreated controls (p > 0.05). The REAC efficacy on spermatozoa oxidative status was also evidenced by the higher trolox equivalent antioxidant capacity (TEAC) found in both the cellular extract (p < 0.05) and the storage media of REAC treated spermatozoa compared to untreated controls (p < 0.0001).ConclusionThe present study demonstrated that REAC treatment during liquid storage preserves spermatozoa acrosome membrane and DNA integrity, likely due to the enhancement of sperm antioxidant defenses. These results open new perspective about the extending of spermatozoa functions in vitro and the clinical management of male infertility.

EFFECTS OF L-CARNITINE ON MORPHOLOGY AND ANTIOXIDANT ENZYMES AND DNA INTEGRITY OF CRYOPRESERVED BUFFALO SPERMATOZOA

Cryopreservation induces sub lethal damage to the spermatozoa, thereby reduce their fertile life. There are some biochemical additives that may enhance buffalo semen freezability; L-carnitine is one of these biochemical semen additives. Till now, the exact effects of L-carnitine on buffalo semen processing outcomes haven’t been discovered. The current study aimed to clarify L-carnitine roles during buffalo semen cryopreservation. Semen was cryopreserved in tris-based extender supplemented with different concentrations of L- carnitine (0.01, 0.05 and 0.1mg/ml)Vs.Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess different semen characteristics. Cryopreserved semen was assessed for percentage post- thawing motility, acrosomal and plasma membrane integrity, viability, DNA damage, antioxidant enzymes concentration, lipid peroxidation, in vitro fertilizing potentials and conception rate. Current results indicated that addition of 0.05 mg/ml L- carnitine to semen extender significantly (P<0.05) improved post-thawing motility, viability and acrosomal integrity (63.33±9.28 %, 133.33±9.40 and 12.33± 2.02 %, respectively) compared with control (43.33±6.01 %, 74.16± 10.93 and 24.67±2.03 %, respectively). Moreover, 0.05mg/ml L- carnitine significantly increased (P<0.05) total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) concentrations (0.50±0.07 mµ/ml, 73.67±5.37 U/ml and 106.66±12.03 U/L, respectively) with respect to the control (0.19±0.01 mµ/ml, 27.33±3.49 U/ml and52.33±4.09 U/L, respectively). Furthermore, 0.05mg/ml L- carnitine significantly decreased (P<0.05) lipid peroxidation of the cryopreserved spermatozoa compared with the control semen (11.00±1.73 vs 24.82±4.90nmol/ml). Likewise, at this concentration sperm DNA damage, tail length and tail moment of the cryopreserved semen significantly (P<0.05) reduced (1.87±0.36 %, 1.83±0.33 µm and 3.72±1.44, respectively) compared with control (3.47±0.13 %, 3.48±0.17 µm and 11.91±0.87, respectively). Additionally, 0.05 mg/ml L- carnitine significantly (P<0.05) improved in vitro fertilization rate (57.45%) and conception rate (61.76%) compared with the control (33.33 and 37.93%, respectively). In conclusion the use of 0.05 mg/ml L-carnitine in the freezing extender improves DNA integrity through enhancing the antioxidant defense of buffalo sperm cells and decreasing the rate of lipid peroxidation. Therefore, L-carnitine may improve sperm cryopreservation quality, reduce cryodamage and improve sperm fertilizing potential

The effects of gelatin supplementation prior to cooling on ram semen quality and fertility.

The physical and chemical characteristics of gelatin have been used to justify its inclusion in extenders to preserve the sperm quality and improve results of cervical artificial insemination with cooled semen. The objective of this study was to evaluate the effects of gelatin supplementation in cooling extender on the quality and fertility of ram semen stored at 5°C. Semen samples (n = 24) of Santa Inês rams (n = 6) were diluted in Glycine-Yolk-Milk extender without (control) or with 1.5% of gelatin. The samples were loaded into 0.25 mL straws, cooled to 5°C and stored vertically for 48 and 72 hours. Sample quality was evaluated using straw homogeneity tests based on pH, osmolality and the proportion of spermatozoa (PS) in both upper and lower segments of straws (US and LS), analyses of sperm motility, plasma and acrosomal membrane integrity, and by fertility after artificial insemination. Differences between the US and LS of straws were found for pH and PS (%). They were significant only in the control group at both times: pH – 5.96 vs. 5.71 at 48 h and 6.13 vs. 5.89 at 72 h; PS – 21.66 vs. 78.34 at 48 h and 20.87 vs. 79.13 at 72 h. Storage in gelatin had very little, to no effect on the sperm kinetics or on the sperm membrane integrity evaluations. The addition of gelatin to the extender did not affect the pregnancy rate which ranged from 4.4 to 26.1%. We conclude that gelatin is effective in maintaining the physical and chemical homogeneity of the semen samples. Further research is needed in order to optimize the use of gelatin supplementation and elucidate any potential benefits.

Cryosurvival of Marwari stallion sperm in different extenders

Extenders (3) were compared for frozen-thawed semen quality of Marwari horses in the present study. Ejaculates (36) including 6 ejaculates from each of 6 stallions were collected using artificial vagina (AV) method. The ejaculates were cryopreserved either with glucose-EDTA-lactose, HF-20 or egg yolk-skim milk extender using custom freezing technique. Sperm characteristics, viz. motility, viability, acrosome intactness and plasma membrane integrity were studied in frozen-thawed semen besides record of fresh semen quality as a prerequisite. Present study indicated a non-significant difference in frozen-thawed semen parameters with glucose-EDTA-lactose and HF-20 extenders, however, both the extenders resulted in a higher percentage of post-thaw motility (PTM), viability, intact acrosome and plasma membrane integrity of sperm than the egg yolk-skim milk extender. It was concluded that glucose-EDTA-lactose and HF-20 are better semen extenders as compared to egg yolk-skim milk and they can be used effectively for semen cryopreservation in horses.

Bull Fertility and Its Relation with Density Gradient Selected Sperm.

Sperm selection method is usually used to collect these cells for in vitro-assisted reproduction. Few studies reported the relationship of in vivo fertility and semen parameters after sperm selection; hence, the present study attempted to assess different semen parameters after post-thaw or sperm selection, using density gradient separation BoviPure®, to predict in vivo fertility. In this experimental study, frozen semen quality of four Montbeliarde bulls were assessed after post-thaw (PT) or after sperm selection (SSp), using density gradient separation BoviPure®, to predict the fertility rate in vivo. In addition to PT or SSp, semen was examined for concentration, motility, morphology abnormalities, viability, acrosome and plasma membrane integrities. Fertility was measured as non-return rates within 56 days after the first insemination (NRR) or as corrected NRR, expressed as CNRR, to the factors influencing fertility using linear mixed model. Non-parametric Kruskal-Wallis test was performed to compare semen parameter variables. Fertility rates were compared using Chi-square test. Pearson correlation analysis was used to test the relationship between CNRR and semen parameters. Data was analysed using SPSS package program, version 21.0. Most of the examined bulls exhibited a high fertility rate (3/4 bulls, 62.1- 81.8% for NRR or 67.2-98.5% for CNRR). Fertility rate, expressed as CNRR, was significantly related to semen parameters after SSp, but not after PT. Thus, CNRR was increased with decrease of total motility, progressive spermatozoa and abaxial implantation frequencies after SSp (r=-0.999, P=0.001; r=-0.990, P=0.010; r=-0.988, P= 0.012, respectively); while, CNRR was decreased with decrease of SSp immotile spermatozoa (r=+0.995, P=0.005), underlying that maximal limit of determined immotile spermatozoa is 47%. High frequencies of total and progressive motility spermatozoa, and abaxial implantation in gradient selected sperm appear to be not favorable for fertility in vivo.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility.

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 106 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro–produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Influence of different anaesthetic protocols over the sperm quality on the fresh, chilled (4°C) and frozen-thawed epididymal sperm samples in domestic dogs.

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n=20), propofol group (PG, n=20) and ketamine-dexmedetomidine group (KDG, n=20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 48hr, and the sperm motility was assessed at 6, 24 and 48hr. In experiment 2, semen samples were diluted in Tris-glucose-egg yolk extender and chilled for 24hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50-100×10(6) spermatozoaml(-1) , 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p<0.05) and progressive sperm motility (p<0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine-dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.

Survival of buffalo bull spermatozoa: effect on structure and function due to alpha-lipoic acid and cholesterol-loaded cyclodextrin

Sperm survival depending upon integral membranes and function is imperative for fertilization. This study was designed to augment survival of buffalo spermatozoa using alpha-lipoic acid (ALA) and cholesterol-loaded cyclodextrin (CLC) during cryopreservation. Semen was frozen using 0, 0.5, 1, 1.5, 2 and 2.5mmolL-1 ALA (experiment 1) and ALA or CLC separately or together (experiment 2). Semen was assessed for post-thaw motility, plasma membrane integrity (PMI), intact acrosome and plasma membrane (IACR-IPM) and DNA integrity at 0, 1.5, 3 and 4.5hr of incubation. In experiment 1, use of 0.5mmolL-1 ALA enhanced the sperm cryosurvival and post-thaw longevity than other groups up to 4.5hr of incubation, and this concentration of ALA was used in second experiment with CLC. The results revealed higher (p<.05) sperm survival function and time of sperm attributes due to use of ALA than CLC and control. However, the sperm quality did not improve (p>.05) when ALA was combined with CLC. In conclusion, survival of buffalo bull spermatozoa during freeze-thawing and post-thaw incubation can be enhanced more with ALA than CLC or control, followed by CLC than control. However, there is no synergistic effect on survival of buffalo bull spermatozoa due to ALA and CLC.

Quality of Fresh, Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.

Efeito de diferentes concentrações de melatonina em espermatozoides de carneiros sobre estresse oxidativo após criopreservação

Resumo: Objetivou-se avaliar o efeito da adição de diferentes concentrações de melatonina no sêmen diluído de carneiros após criopreservação. Foram coletados 10 ejaculados de três carneiros adultos (n=30), por meio de vagina artificial para ovinos. Os ejaculados coletados foram diluídos em Tris-Gema de ovo, para a concentração final de 200x106 sptz/mL, mantidos em banho maria a 32°C, e a melatonina adicionada conforme os tratamentos: Controle; 100pM; 100nM; 100μM e 1mM de melatonina. Então, as amostras foram resfriadas em câmara fria a 5°C por duas horas, envasadas em palhetas de 0,5 mL e lacradas. Logo após, foram acondicionadas sob vapores do nitrogênio liquido, por 15 minutos, a 8cm da lâmina líquida e congeladas com nitrogênio líquido. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática, membrana acrossomal, atividade mitocondrial, quantificação do estresse oxidativo e a capacidade de ligação. As variáveis foram submetidas à análise de variância e as médias foram comparadas pelo teste de Tukey a 5% de probabilidade. A motilidade total e progressiva dos espermatozoides descongelados foi maior nas amostras tratadas com 100pM de melatonina (62,99 e 45,07% respectivamente; P&lt;0,05) quando comparado aos demais tratamentos. A adição das diferentes concentrações de melatonina no sêmen diluído, com exceção da concentração de 1 mM, apresentou maior percentual de células com membrana plasmática íntegra, quando comparadas com o controle (P&lt;0,05). O percentual de espermatozoides com integridade da membrana do acrossoma foi maior no sêmen tratado com 100 pM de melatonina (P&lt;0,05) do que nos demais tratamentos. A alta atividade mitocondrial foi maior nos espermatozoides tratados com 100 pM de melatonina (69,30%; P&lt;0,05). A adição de 100 nM de melatonina reduziu a quantidade de TBARS após a criopreservação (2,84; P&lt;0,05) quando comparado aos demais tratamentos. Após o descongelamento, o número de espermatozoides que se ligaram à membrana perivitelina foi maior nos tratados com 100 pM de melatonina (155,73; P&lt;0,05). Portanto, a adição de melatonina no sêmen diluído pode ser útil para aperfeiçoar a criopreservação do sêmen de ovinos, melhorando as taxas de fertilização por meio da inseminação artificial.

Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion), scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot) and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342). The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

Effect of different extenders on recovery and storage of epididymal ram spermatozoa

This experiment was conducted to determine effect of different extender on recovery and storage of epididymal ram spermatozoa. Testes were obtained from eight mature rams which were slaughtered at four different occasions, a pair in each session. Caudal epididymis was isolated and divided to four equal parts. Spermatozoa recovery from each part of the caudal epididymis was performed by milk (M), Tris (T), Tris plus caproic acid (T-C) and sodium citrate (S) extenders. Spermatozoa in each extender were divided into four parts and then the amount of 0 (E0), 5 (E5), 10 (E10) and 15% (E15) egg yolk were added. Samples were chilled to 4 °C and stored for 7 days. The evaluation of sperm motility, viability, membrane integrity and acrosome integrity were performed daily. The results showed that there was interaction between egg yolk and storage time and also egg yolk and extender on sperm motility (p < 0.05). Sperm motility was highest at 15 % egg yolk (25.6 %) on 7 d (p < 0.05). Sperm motility was higher in ME15 (57.8 %) and T-CE15 (62.3 %) than TE15 (53.4 %) and SE15 (41.9 %; p < 0.05). There was interaction among egg yolk, extender and storage time on acrosome integrity, membrane integrity and sperm viability (p < 0.05). On 7 d, there were no differences among ME15, T-CE15 and TE15 on sperm viability and membrane integrity. Acrosome integrity was higher in T-CE15 (75.25 %) than ME15 (62.5 %) on 7 d (p < 0.05). Therefore, Tris caproic extender was superior to other extender for recovery and storage of ram epididymal spermatozoa.

In vitro fertilization using frozen-thawed feline epididymal spermatozoa from corpus and cauda regions

Epididymal sperm preservation offers a potential for rescuing genetic material from endangered or valuable animals after injury or death. Spermatozoa from corpus, as well as from cauda, have the capability to be motile and to undergo capacitation and can thus potentially be preserved for assisted reproductive technologies. In the present study, feline frozen-thawed epididymal spermatozoa from corpus and cauda regions were investigated for their ability to fertilize homologous oocytes and further embryo development in vitro. Epididymal spermatozoa from corpus and cauda of seven cats were cryopreserved and used for IVF. Cumulus–oocyte complexes (n = 419) were obtained from female cats after routine spaying. Frozen-thawed corpus epididymal spermatozoa showed similar properties of acrosome integrity, membrane integrity, and chromatin integrity as frozen-thawed spermatozoa from cauda except corpus spermatozoa showed lower motility (P < 0.05). The fertilizing capacity of frozen-thawed corpus epididymal spermatozoa was confirmed by similar number of embryos developing to the two- and four-cell stages compared with sperm from cauda (32.03% vs. 33.33%). However, oocytes fertilized with corpus spermatozoa had lower potential to develop to the blastocyst stage (6.79%) and had lower cell numbers compared to oocytes fertilized with cauda spermatozoa (14.08%). In conclusion, spermatozoa from corpus epididymis had a similar capability to fertilize homologous oocytes in vitro as sperm from cauda but resulted in fewer embryos developing to the blastocyst stage compared to spermatozoa from the cauda.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing1,2

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Quality decline and oxidative damage in sperm of freshwater crab Sinopotamon henanense exposed to lead

Lead (Pb) induces male infertility in vertebrates. Whether lead is related to reproductive abnormalities in aquatic invertebrates remains uncertain. In this work, effects of Pb on the sperm quality and oxidative stress of the freshwater crab Sinopotamon henanense were investigated after 3, 5 and 7d exposure to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L). Sperm quality indices including sperm plasma-membrane integrity and acrosomal-membrane integrity were measured by flow cytometry. DNA integrity was measured by fluorescence microscopy. The results showed that Pb levels in sperm increased significantly upon Pb exposure in most treated groups, sperm plasma-membrane integrity, acrosomal-membrane integrity, and DNA integrity were reduced at higher concentrations after 5 d and 7d. Oxidative stress of sperm induced by Pb was reflected in significant up-regulation of reactive oxygen species (ROS) levels after 3, 5 and 7d. A significant reduction of the total antioxidant capacity levels occurred after exposure to 14.7mg/L Pb and above at 7d compared to the control. The results of oxidative damage to lipids, proteins and DNA of sperm showed that malondialdehyde, protein carbonylation and DNA–protein crosslinks were increased in a concentration- and time-dependent manner. Our findings document that Pb can induce harmful effects on several reproductive endpoints in a freshwater crab.