Membrane Acrosome Integrity Research Articles

Effects of Degasified Extender on Quality Parameters of Cryopreserved Bull Spermatozoa

One of the most important factors contributing to poor quality semen post thaw has been reported to be oxidative stress. Continued interaction of spermatozoa with dissolved oxygen in extender renders detrimental effect by generating reactive oxygen species during processing. This formed the basis to investigate the effects of removal of oxygen from the Tris-Glycerol-Egg Yolk (TG-EY) extender on the post-thaw semen quality. Semen ejaculates (n=26) collected from three cross bred bulls with mass activity of ≥3 and progressive motility of ≥70% were split in two fractions (control and treatment) to obtain 80 x 106 progressively motile sperm/mL of extended semen. The TG-EY extender composed of 3.028 g of Tris, 1.675 g of citric acid, 1.25 g of fructose, 7.0 mL of glycerol, 10 mL of egg yolk, 105 IU Penicillin G Sodium and 105 µg Streptomycin for 100 mL of deionized water for control and degasified (freeze-pump-thaw cycling method using liquid nitrogen) for treatment. Semen was processed and filled in 0.25 mL polyvinyl chloride straws, sealed and cryopreserved for at least 2 week before evaluation. Assessment of post-thaw motility, viability, acrosome and plasma membrane integrity of spermatozoa were evaluated after thawing at 37°C for 30s. Study revealed a significant (viability and acrosome integrity, p

Protein profile and functionality of spermatozoa from two semen collection methods in Bali bulls

The aim of this study was to evaluate the effect of two semen collection methods (electro-ejaculation [EE] and transrectal massage [RM]) on in vitro sperm functionality and protein composition of seminal plasma in Bali bulls. Ten untrained Bali bulls were selected for semen collection by EE and RM. Parameters analysed were acrosome and plasma membrane integrity, sperm motility (by CASA), normal morphology, functionality (sperm penetration assay), acrosome reaction, total protein content and protein profiles (by 2D-PAGE). Bulls collected by RM had a higher (p<0.05) percentage of spermatozoa with intact acrosome and plasma membrane, functionality and individual motility, and a lower proportion of seminal plasma, total protein content and lower ratio of low molecular weight proteins than those collected by EE. Analysis of 2D-PAGE gel detected about 116 spots in the range of 10–250kDa and isoelectric points (pI) ranging from 3 to 10. Approximately 52% of seminal plasma protein spots were represented by four major protein fractions with molecular weights around 37–45kDa (15.66%), 25–30kDa (12.46%), 14–16kDa (11.73%) and 12–15kDa (11.52%). Ten of the seminal plasma proteins identified by mass spectrometry belonged to major bovine seminal plasma proteins. A very significant finding in this study was related to the two proteins identified, PGK and PLA2, with MW of approximately 37–40 and 50–55kDa and pI of 8.5–8.8 and 5.2–6.0, respectively. These two protein spots can only be detected in the seminal plasma of ejaculates obtained through RM. In conclusion, semen quality as examined by in vitro sperm functionality was found to be better in RM than EE samples after treatment with heparin and calcium ionophore A-23187. In addition some low molecular weight proteins were up-regulated in the seminal plasma obtained from the EE method.

265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P &lt; 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P &lt; 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P &lt; 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P &lt; 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.

64 COULD FROZEN–THAWED BOAR-SEMEN FERTILITY BE INCREASED WITH SEMINAL PLASMA ADDITION?

Post-thawed addition of seminal plasma (SP) in equine cryopreserved semen increased the integrity of plasma and acrosomal membranes (Andrade et al. 2011 Reprod. Dom. Anim. 46, 682–686) and these possibly affect sperm lifespan, improving fertility (Garcia et al. 2010 Anim. Reprod. Sci. 119, 160–165). This study was conducted to verify the pregnancy (PR) and fertility rate (FR) of addition of homologous SP in thawed boar semen. First, SP collections were made with polled sperm rich-fraction. Semen was centrifuged (500 × g for 10 min) and supernatant was removed, centrifuged one more time (2500 × g for 30 min), vacuum filtered through membranes (0.22 μm), and stored at –80°C for future use. Samples collected to frozen were pooled and divided in 2 aliquots, control (cryopreserved with SP; CON) and cryopreserved without SP (WSP; SP was removed and discarded after centrifugation – 500 × g for 10 min). Samples were extended in freezing extender (Botupharma®) to a final concentration of 300 × 106 spermatozoa per milliliter, packaged in 0.5-mL straws, and frozen in an automatic system (TK Tecnologia em Congelação®) using a rate of –0.5°C min–1 until 5°C, –20°C min–1 until –120°C, and then immersed in LN (–196°C). Ten straws, from each treatment, were thawed in water bath (37°C/30 s) and extended in Beltsville thawing solution to obtain 1.5 × 109 sperm in 40 mL. The other 10 straws of WSP were thawed and extended in Beltsville thawing solution plus 10% (v:v) of SP to originate treatment TSP (thawed added of seminal plasma). Thirty-three (11 per treatment) gilts had synchronized ovulation with altrenogest (25 mg/5 mL, Regumate®) administration per 18 days. Following day after withdrawal the altrenogest was administered with an intramuscular injection of 600 IU of eCG (Novormon®) and 2.5 mg of pLH (Lutropin®-V) after 72 h; a single deep intrauterine insemination was made 36 h after. Five days after, females were slaughtered and embryos were collected (by oviducts flushed) and evaluated by esteromicroscopia. Fertility rate and PR were analysed by SAS program (SAS Institute Inc., 2010, Cary, NC, USA). Fertility rate was analysed by Mixed models, and treatment effects were analysed by orthogonal contrasts (C1: effect without SP = CW v. NC; C2: effect of post-thawed addition of SP = CP v. CW), and PR was evaluated by binary distribution with PROC GLIMMIX test. Fertility rate was not affected (P &gt; 0.05) by cryopreservation of boar semen in presence or absence of SP nor by its addition in Beltsville thawing solution (10.58 ± 3.92, 9.57 ± 4.92, 21.29 ± 7.37 for CON, WSP, and TSP, respectively). Treatments did not influence (P &gt; 0.05) PR (50.00, 36.36, 72.73 for CON, WSP, and TSP, respectively). Thus, neither SP addition in thawed boar semen nor cryopreservation with or without SP can be beneficial to PR and FR, in our experimental conditions. However, a numeric large difference was observed. Therefore, these results lead us to believe that SP have a potential to increase the fertility and pregnancy rate, and that can be verified in further studies, with more repetitions.Research was supported by Agroceres Multimix, Botupharma and FAPESP process 2013/08070-8 and 2011/23484-8.

Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm

Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80μM, 120μM, 160μM, 200μM, 240μM, or 320μM) for 45min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160μM Hoechst33342 for various durations (0min, 15min, 30min, 45min, 60min, 75min, or 90min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160μM. Moreover, when the staining duration was equal to or longer than 45min, the frozen-thawed sperm can be successfully sorted in the presence of 160μM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro fertilizing capability. Accordingly, the minimum values of Hoechst33342 concentration and staining duration can be set at 160μM and 45min respectively. However, the maximum values of Hoechst33342 concentration and staining duration were not determined based on the current study. Further research on how to reduce injuries caused by freezing, thawing, and Hoechst33342 staining on frozen-thawed ram sperm is needed.

Antioxidant effect of lemon balm (Melissa officinalis) and mate tea (Ilex paraguensys) on quality, lipid peroxidation and DNA oxidation of cryopreserved boar epididymal spermatozoa.

In this study, we investigated the protective ability of the addition of two antioxidant herb extracts, mate tea and lemon balm, on boar epididymal frozen-thawed spermatozoa quality. Testes from mature boars were collected at local slaughterhouse, and sperm samples from epididymis were recovered by flushing. Spermatozoa were cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of lemon balm and mate tea (0, 2.5, 5 and 10 g l(-1) ) using the straw-freezing procedure. Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels and DNA oxidative damage (8-hydroxy-2'-deoxyguanosine base lesion) were evaluated. There were no differences among experimental groups with regard to motility characteristics, viability, acrosome and plasma membrane integrity; however, the highest concentration of lemon balm produced significant (P < 0.05) improvement in curvilinear trajectory, straightness and amplitude of lateral head displacement after thawing. The supplementation of freezing extender with mate tea and lemon balm reduced sperm lipid membrane peroxidation, and only mate tea protected DNA against oxidative damage during cryopreservation at 120 min post-thawing (P < 0.05). Mate tea experimental extender at concentration of 10 g l(-1) showed the lowest percentage of sperm oxidised DNA and malondialdehyde generation; thus, mate tea is a potential candidate such as antioxidant compound on boar sperm cryopreservation medium.

Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance

The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA.

AMPK up-activation reduces motility and regulates other functions of boar spermatozoa.

We recently demonstrated that AMPK inhibition in spermatozoa regulates motility, plasma membrane organization, acrosome integrity and mitochondrial membrane potential. As AMPK activity varies in different energy conditions induced by sperm environment, this work investigates the functional effects of AMPK activation in boar spermatozoa. Spermatozoa were incubated under non-stimulating (TBM) or Ca(2+) and [Formula: see text]-stimulating (TCM) media in the presence/absence of AMPK activator, A769662, for different times. AMPK activity, evaluated as Thr(172) phosphorylation by western blot, is effectively increased by A769662 in spermatozoa. AMPK activation significantly reduces the percentage of motile spermatozoa under Ca(2+) and/or [Formula: see text]-stimulating conditions. Moreover, AMPK activation in spermatozoa incubated in TBM or TCM significantly reduces curvilinear VCL, straight-line VSL and average VAP velocities, which subsequently lead to a significant decrease in the percentage of rapid spermatozoa (VAP > 80 μm/s). The effect of AMPK activation on motility is intensified by the absence of BSA in the incubation medium. AMPK activation for a short time prevents the decline in cell viability and in the sperm population displaying high mitochondrial membrane potential which is induced by Ca(2+) and [Formula: see text]. Sustained (24 h) AMPK activation under TBM or TCM significantly increases both lipid disorganization and phosphatidylserine externalization in the sperm plasma membrane, and diminishes the acrosome membrane integrity. In summary, AMPK activation modifies essential sperm processes such as motility, viability, mitochondrial membrane potential, acrosome membrane integrity, and organization and fluidity of plasma membrane. As these spermatozoa processes are required under different environmental conditions when transiting through the female reproductive tract to achieve fertilization, we conclude that balanced levels of AMPK activity are essential for regulating sperm function.

Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa

The cryopreservation of dromedary camel (Camelus dromedarius) sperm has proved challenging with little success reported. The routine application of artificial insemination with frozen semen would assist the flow of valuable genetic material nationally and internationally. The current study sought to examine the effects of cholesterol (cholesterol-loaded cyclodextrin [CLC]) preloading on camel sperm cryosurvival. Ejaculates (n = 3 males; 3 ejaculates per male) were collected using an artificial vagina during the breeding season and extended in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) and allowed to liquefy in the presence of papain (0.1 mg/mL) before removal of the seminal plasma by centrifugation. Sperm pellets were resuspended (120 million/mL) in fresh TALP and incubated (15 minutes; 37 °C) with 0, 1.5, or 4.5 mg CLC/mL. Sperm suspensions were then centrifuged and reconstituted in INRA-96 containing 20% (v:v) egg yolk and 2.5% (v:v) methylformamide, loaded in 0.5-mL plastic straws, sealed, and cooled for 20 minutes at 4 °C. Straws were frozen over liquid nitrogen (4 cm above liquid; 15 minutes), plunged, and stored. Sperm motility, forward progressive status, and acrosomal integrity were recorded at 0 and 3 hours after thawing and compared with these same parameters before freezing. Aliquots also were stained with chlortetracycline hydrochloride to assess spontaneous sperm capacitation status before freezing and post-thaw. Pretreatment with CLC (1.5 and 4.5 mg/mL) enhanced cryosurvival. Post-thaw sperm motility was highest (P < 0.05) in 1.5 mg CLC/mL immediately after thawing (44%) and after 3 hours incubation at room temperature (34%). Highest post-thaw sperm progressive status was also achieved in the presence of 1.5 CLC. Greater proportions of spermatozoa retained acrosomal membrane integrity when cryopreserved in the presence of CLC, but there was no difference between 1.5 and 4.5 CLC. Although thawed spermatozoa underwent spontaneous capacitation during in vitro incubation, cryopreservation and CLC treatment exerted no effect. In summary, dromedary camel sperm benefit from exposure to CLC before cryopreservation; this may facilitate the routine collection and storage of sperm from this species.

Eficiência dos diluidores Tris e Botu-crio® sobre os parâmetros seminais de garanhões das raças quarto de milha e mangalarga marchador

O principal fator a limitar o processo de criopreservação de sêmen equino está relacionado à própria espécie, por apresentar uma grande variabilidade nas características dos ejaculados pós-descongelação. O objetivo desta pesquisa foi avaliar a viabilidade espermática pós-descongelação, do sêmen de garanhões das raças Quarto de Milha e Mangalarga Marchador utilizando-se dois diluentes (Tris e Botu-crio®) na criopreservação. Para tanto, foram analisadas as características físicas do sêmen fresco, funcionalidade da membrana espermática pelo Teste Hiposmótico (HOST), motilidade total e vigor pelo Teste de Termoresistência (TTR), motilidade progressiva pelo sistema computadorizado CASA (Computer Assisted Sêmen Anlyses) e integridade da membrana espermática, do acrossoma e funcionalidade de mitocôndria por microscopia de epifluorescência. O diluente Botu-crio® preservou melhor os valores de motilidade e vigor e a integridade de membrana plasmática, pós-descongelamento. Não houve diferença significativa entre as raças quanto à motilidade total e vigor no sêmen fresco, bem como para a análise desses parâmetros no Teste de Termoresistência pós-descongelamento. Garanhões da raça Quarto de Milha apresentaram percentual maior de defeitos maiores na análise de patologia espermática.

Criopreservação do sêmen ovino em meio diluente à base de água de coco em pó (ACP-102c)

O objetivo deste trabalho foi avaliar o diluente ACP-102c na criopreservação do sêmen ovino em comparação com o diluidor tris-glicose-gema (TRIS) e o sêmen fresco. Foram coletados 48 ejaculados de quatro ovinos, sendo tomadas duas alíquotas por ejaculado para diluição e criopreservação em ACP-102c ou TRIS e uma terceira alíquota utilizada para análise do sêmen fresco. O sêmen fresco e o criopreservado em ambos os diluidores foram avaliados para viabilidade, integridade de membrana plasmática e acrossomal, teste hiposmótico, fragmentação do DNA e de motilidade espermática. Após descongelamento, ambos os diluidores não diferiram para viabilidade espermática, integridade de membrana plasmática e acrossomal, fragmentação de DNA e nas variáveis quantitativas e qualitativas de velocidade espermática, mas diferiram no teste hiposmótico, motilidade total e progressiva e amplitude lateral da cabeça, bem como em todas as variáveis de motilidade avaliadas, exceto linearidade e progressividade, após duas horas de incubação à 37 ºC. Houve variabilidade entre reprodutores na motilidade total e progressiva do sêmen criopreservado em ACP-102c após descongelamento. O diluidor ACP-102c conferiu menor proteção aos espermatozoides ovinos contra danos do congelamento quando comparado ao TRIS, mas o aprimoramento de sua formulação e protocolos mais adequados de congelação poderão torná-lo uma alternativa na congelação do sêmen ovino.

Rosmarinic acid improves function and in vitro fertilising ability of boar sperm after cryopreservation

During cryopreservation, oxidative stress exerts physical and chemical changes on sperm functionality. In the present study we investigated the antioxidant effect of rosmarinic acid (RA) on quality and fertilising ability of frozen–thawed boar spermatozoa. Ejaculates collected from mature boar were cryopreserved in lactose–egg yolk buffer supplemented with different concentrations of RA (0μM, 26.25μM, 52.5μM and 105μM). Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels, DNA oxidative damage (8-hydroxy-2-deoxyguanosine base lesion) and in vitro fertilisation ability were evaluated. Total and progressive motility were significantly higher in experimental extenders with RA than in the control (P<0.05) at 0 and 120min post-thawing. The plasma and acrosomal membrane integrity were improved by supplementation with 105μMRA (P<0.05). Negative correlation between RA and malondialdehyde (MDA) concentration were determined (P<0.05). After thawing, the percentage of spermatozoa with oxidised DNA did not differ between extenders, however, at 120 and 240min post-thawing, the samples supplemented with 105μMRA showed the lowest DNA oxidation rate (P<0.05). The penetration rate was significantly higher on spermatozoa cryopreserved with 105μMRA (P<0.05). The results suggest that RA provides a protection for boar spermatozoa against oxidative stress during cryopreservation by their antioxidant properties.

Effect of conventional and controlled freezing method on the post thaw characteristics of boar spermatozoa

The objective of the present study was to evaluate the effectiveness of conventional, and controlled freezing method adopting three freezing rates 20°C, 40°C and 60°C/min for cryopreservation of boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender using 0.5ml straws. Semen samples were evaluated for sperm motility, live sperm, acrosome integrity, plasma membrane integrity (PMI) by carboxyfluorescein diacetate plus propidium iodide (PI) staining, mitochondrial membrane potential (MMP) by combined JC-1 plus PI staining and lipid peroxidation (LPO) by BODIPY 581/591-C11 probe after equilibration and after freezing. The results revealed that the post thaw sperm motility, live sperm, live intact acrosome and plasma membrane integrity were significantly (p<0.05) higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the controlled freezing methods yielded higher (p>0.05) mean values of live sperm with high MMP as compared to conventional freezing. However, the post thaw sperm LPO did not influence by difference in freezing methods. No significant difference on the post thaw sperm qualities was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly (p<0.05) after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, cryopreservation of boar semen with controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes

Fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were used to simultaneously evaluate the integrity of plasma and acrosomal membranes as well as mitochondrial membrane function in cryopreserved bovine semen and to verify its influence on fertility and postinsemination uterine vascularization. One hundred eighty-two Nellore cows were distributed for artificial insemination (AI) using semen batches separated according to the cell percentage presenting intact plasma membrane, intact acrosome, and high mitochondrial function (IPIAH): group G (44.5% IPIAH, n = 68), group M (23.0% IPIAH, n = 56), and group R (8.5% IPIAH, n = 58). The uterine hemodynamic was evaluated by Doppler sonogram in three periods: 30 hours before AI, 4 and 24 hours after AI were considered the resistance index and the uterine vascularization score. The pregnancy rate of group G (64.7%) was greater (P > 0.05) compared with group R (36.2%), but both did not differ from group M (50.0%). There was no effect (P > 0.05) of semen quality on uterine vascularization. Greater vascularization was noticed 4 hours after AI than 30 hours before and 24 hours after AI. Semen evaluation using fluorescent probes contributes to predicting fertilizing potential of semen. The use of semen with less percentage of IPIAH sperm does not alter uterine hemodynamic in cows.

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000mOsm), sucrose (369 and 479mOsm), 1M glycerol (1086mOsm) or dimethyl sulfoxide (Me2SO, 1151mOsm) for 10min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P>0.05), NaCl and sucrose solutions affected sperm motility (P<0.05), but not membrane integrity. Motility of sperm exposed to <600mOsm NaCl or sucrose was less (P<0.05) than fresh ejaculate, but comparable (P>0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1M glycerol or Me2SO and cooled from 5°C to −120°C at −57.8°C, −124.2°C or −67.0°C/min, frozen in LN2, thawed in a water bath for 30s at 37°C or 10s at 50°C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P<0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P<0.05) post-thaw motility (20.0±1.9% versus 13.5±2.1%) and membrane integrity (51.2±1.7% versus 41.5±2.2%) were observed in samples cryopreserved in Me2SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8°C/min being advantageous (P<0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me2SO as a cryoprotectant.

Effect of Superoxide Dismutase on Semen Parameters and Antioxidant Enzyme Activities of Liquid Stored (5°C) Mithun (Bos frontalis) Semen

The present study was undertaken to assess the effect of superoxide dismutase (SOD) on sperm motility, and viability; total sperm abnormality; acrosomal and plasma membrane integrity; DNA abnormality; antioxidant profiles such as catalase (CAT), reduced glutathione (GSH), and total antioxidant capacity (TAC); enzymatic profiles such as aspartate amino transaminase (AST), and alanine amino transaminase (ALT); and biochemical profiles such as malondialdehyde (MDA) production and cholesterol efflux. Total numbers of 50 ejaculates were collected twice a week from eight mithun bulls and semen was split into four equal aliquots, diluted with the TEYC extender. Group 1: semen without additives (control), and group 2 to group 4: semen was diluted with 50 U/mL, 100 U/mL, and 150 U/mL of SOD, respectively. These seminal parameters, antioxidant, enzymatic, and biochemical profiles were assessed at 5°C for 1, 6, 12, 24, and 30 h of incubation. Inclusion of SOD into diluent resulted in significant (P&lt;0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa, and acrosomal abnormalities at different hours of storage periods as compared with control group. Additionally, SOD at 100 U/mL has significant improvement in quality of mithun semen than SOD at 50 or 150 U/mL stored inin-vitrofor up to 30 h. It was concluded that the possible protective effects of SOD on sperm parameters are that it prevents MDA production and preserves the antioxidants and intracellular enzymes during preservation.

Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state

Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5°C for 48h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov® (BB)] and another composed of 1% soy lecithin [Botu-Bov®-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/108 cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n=253) or semen cooled for 48h in TRIS-R (n=233), BB (n=247) or BB-L (n=240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-h of the refrigeration period (P<0.01), BB-L conferred greater protection against oxidative stress (P<0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRIS-C, TRIS-R, BB and BB-L were 39.92a, 25.32b, 26.32b and 33.33ab, respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48-h period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen.

Organic selenium increases PHGPx, but does not affect quality sperm in raw boar semen

This study assessed the effects of feeding organic selenium (Se) on sperm morphology, motility, membrane integrity and lipidic peroxidation, also on sperm ATP and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in raw semen of mature boars. Twelve boars were divided into three groups: Control, 0.3ppm sodium selenite, Inorganic, 0.5ppm sodium selenite and Organic, 0.5ppm Se yeast for 11 weeks. Organic diet presented a higher total sperm number (P<0.05) when compared to the inorganic treatment; however, no difference was observed regarding volume, sperm concentration, number of cells with plasma and/or acrosomal membrane integrity, membrane mitochondrial potential, ATP assay and Se concentration in seminal and blood content. Also, no difference was observed on motility parameters, sperm morphology and membrane peroxidation, however, PHGPx was influenced (P<0.05) in organic treatment. Thus organic Se feeding influenced none of the analyzed seminal characteristics in raw semen, except for PHGPx and total number of sperm.

Changes in motility, morphology, plasma membrane and acrosome integrity during stages of cryopreservation of buck sperm.

Changes in sperm structure and function occur during the processing of semen. The present study was designed to investigate the effect on buck sperm during different stages of semen preparation including dilution, cooling, equilibration and freeze-thawing. Semen ejaculates from three mature bucks (replicates = 5) were diluted with tris-citric acid egg yolk glycerol extender at 37 ºC, cooled to 4 ºC over 90 min, equilibrated at 4 ºC for 2 h, transferred to 0.5 mL straws, placed in nitrogen vapour, frozen and thawed and then analysed. Sperm samples were assessed for percentage motility, acrosomal and plasma membrane integrity, live sperm, and morphology after dilution, cooling, equilibration and thawing. Mean percentage motility after dilution (86.0 ± 1.4%) was reduced significantly (p &lt; 0.05) due to cooling and equilibration (77.6 ± 1.3% and 74.6 ± 1.4% respectively); furthermore, it decreased significantly (p &lt; 0.05) after freezing and thawing (42.3 ± 2.5%). Mean percentage of live sperm was higher (p &lt; 0.05) after dilution (89.3 ± 1.4%)compared with cooling (84.8 ± 1.8%) and equilibration (80.2 ± 2.5%) and further reduced (p &lt; 0.05) after freezing and thawing (56.0 ± 3.4%). Sperm morphology dropped significantly (p &lt; 0.05) from 96.4 ± 0.3% after dilution to 88.8 ± 1.3% at cooling and further decreased (p &lt; 0.05) after freezing and thawing (81 ± 1.9%). Mean percentage of sperm with normal plasma membrane after dilution (82.2 ± 1.1%) was significantly reduced (p &lt; 0.05) at cooling or equilibration (73.8 ± 1.8) and further decreased (p &lt; 0.05) after freezing and thawing (50.1 ± 2.9%). The percentage of sperm with normal acrosomes did not differ significantly due to dilution, cooling or equilibration (85.8 ± 1.7%, 83.2 ± 1.6%, 81.7 ± 1.8%) but was significantly reduced after freezing and thawing (45.2 ± 2.8%). In conclusion, frozen thawed sperm showed maximum damage to motility, morphology, plasma membrane and acrosome integrity following cooling.

Daily exposure to silver nanoparticles during prepubertal development decreases adult sperm and reproductive parameters

As silver nanoparticles (AgNPs) have antimicrobial properties and potentiate the activity of some antibiotics, they are broadly used in both medical and nonmedical applications. In this study, prepubertal male Wistar rats were orally treated with 15 or 30 µg/kg/day AgNPs from postnatal day 23 (PND23) to PND58 and sacrificed at PND102. The acrosome integrity, plasma membrane integrity, mitochondrial activity and morphological alterations of the sperm were analyzed. Sexual partner preference, sexual behavior and the serum concentrations of FSH, LH, testosterone and estradiol were also recorded. The results were evaluated following the appropriate statistical analyses, and differences among the groups were considered significant when p < 0.05. AgNPs reduced the acrosome and plasma membrane integrities, reduced the mitochondrial activity and increased the abnormalities of the sperm in both treatment groups. AgNP exposure also delayed the onset of puberty, although no changes in body growth were observed in either treatment group. The animals did not show changes in sexual behavior or serum hormone concentrations. This study shows for the first time that prepubertal exposure to AgNPs causes alterations in adult sperm parameters. Importantly, the sperm appeared to be more sensitive to the toxic effects of AgNPs and demonstrated adverse effects following exposure to lower doses. Consequently, the effects of AgNPs on sperm should be considered in order to establish safety limits for the use of these particles.