Impact of seminal plasma superoxide dismutase and glutathione peroxidase on cryopreserved buffalo spermatozoa.

Fifty semen samples were collected from sixteen buffalo-bulls (4-10 years old) and evaluated before cryopreservation. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) as well as the levels of glutathione (GSH) and malondialdehyde (MDA) were assayed in the seminal plasma before freezing. Aspartate aminotransferase (AST) activity and cholesterol content were assayed in seminal plasma before freezing and after thawing. Results revealed the presence of SOD and GPx activities (0.07 ± 0.01 U/ml and 14.59 ± 0.50 nmol/min/ml, respectively) in buffaloes' seminal plasma. SOD activity was positively correlated with both of GSH level and GST activity in seminal plasma, and showed an inverse relationship with both cholesterol efflux and post-thaw abnormal tails of buffalo spermatozoa. A positive correlation was found between GPx activity in seminal plasma and abnormal tails and an inverse relationship with both post-thaw viability indices and increased motility in response to PTx. GST activity showed a positive correlation with the increased motility after addition of PTx and negative correlations with both of cholesterol level and AST activity. MDA levels were negatively correlated with motility after addition of PTx and positive correlations with both post-thaw abnormal acrosomes and tails. Buffalo seminal plasma contains high activities of SOD, GPx and GST enzymes and GSH levels that have an influence on the functional competence of cryopreserved spermatozoa.

Selectivity of porcine zona pellucida to bind spermatozoa with normal chromatin structure.

This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of binding capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process.

Effect of ram age on structural and functional competence of frozen-thawed spermatozoa in dairy sheep.

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen-thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1-2 years (young) or 4-5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of >or= 2.5 x 10(9) sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY(581/591), sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = -0.63 to -0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.

Sperm chromatin stability during in vitro manipulation of beef bull semen.

Seven experiments were conducted to study the effect of freezing extenders, antioxidants, motility stimulants, thawing temperature, incubation temperature and time, centrifugation and capacitation on sperm chromatin instability (CI) as well as the influence of sperm CI on pregnancy rates of heifers (n = 360) after AI with frozen semen. Semen was collected once a week from Blonde d'Aquitaine and Limousine bulls (n = 3/breed) via an artificial vagina and only individual ejaculates (n = 300) of >0.3 x 10(9) sperm/ml and >or= 70% progressive motility were used. Sperm CI was evaluated by nuclear DNA susceptibility to acid-induced denaturation using acridine orange fluorescence and by chromatin susceptibility to decondensation using quantitative transmission electron microscopy. Bioxcell extender was better than AndroMed and egg yolk extenders in terms of low incidence of sperm CI in one bull (p < 0.05). Neither antioxidants (EDTA-2Na, Na-pyruvate and albumin) nor motility stimulants (caffeine and blood serum) had any significant effect on sperm CI. Thawing of frozen semen at 45 degrees C for 30 s decreased (p < 0.025) CI in one bull. Incubation of frozen sperm at 25 and 39 degrees C for 240 min increased sperm CI percentages from 3.47 +/- 0.48 and 4.50 +/- 0.41% to 6.70 +/- 0.36 and 9.71 +/- 0.53%, respectively (p < 0.001). Although centrifugation and removal of extracellular milieu increased CI of cooled sperm, it decreased CI of frozen-thawed sperm (p < 0.025). Follicular fluid as a capacitating agent destabilized chromatin structure (p < 0.001). Sperm vulnerability to CI had a negative impact (r(2) = 0.37-0.77, p < 0.001) on fertility of frozen ejaculates. In conclusion, in vitro manipulation of bovine semen can influence incidence of sperm CI, whereas integrity of sperm chromatin contributes significantly to heifers' fertility. We would recommend selection of the appropriate extender and thawing temperature for each bull together with careful manipulation of frozen semen to minimize damage of sperm chromatin.

Testing usability of butylated hydroxytoluene in conservation of goat semen.

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled-stored semen as well as motility and kidding rate of frozen-thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk-based and egg yolk-free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mM BHT and stored at 5 degrees C for 168 h. In the third experiment, 30 ejaculates were collected from the above-mentioned bucks, split and diluted with egg yolk-free extenders supplemented with or without 0.3, 0.6 and 0.9 mM BHT and egg yolk-based extenders supplemented with or without 5 mM BHT. Diluted semen was cooled to 5 degrees C over a period of 4 h, frozen and thawed in the form of 0.3-ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk-free extenders containing 0.6 mm BHT and egg yolk-based extenders supplemented with or without 5 mM BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120-140 x 10(6) progressively motile spermatozoa was used for a single cervical insemination of cloprostenol-synchronized does (n = 230). The results showed that addition of 5 mM BHT to egg yolk-deficient (2.5%) extenders significantly improved viability of chilled-stored semen together with motility (48.5%) and fertility (62.5%) of frozen-thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled-stored semen but also post-thaw motility (47.5%) and fertility (53.75%) of frozen-thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.

Factors affecting chromatin stability of bovine spermatozoa.

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

Pellet-freezing of Damascus goat semen in a chemically defined extender.

During the breeding season of goats (12 bucks and 64 does) in Egypt, five experiments were conducted using a chemically defined cryoextender (CDE) to investigate: (1) the influence of rates of semen dilution (1:2, 1:4 and 1:19) and methods of thawing of frozen semen pellets (dry thawing versus wet thawing) on sperm progressive motility (SPM), sperm acrosome abnormalities (SAA) and rate of lipid peroxidation in semen as measured by malonaldehyde (MAL) production, and (2) the effect of insemination of does in natural (n = 38) and cloprostenol-synchronized (n = 26) estrus with frozen semen on their kidding rates and prolificacy. Semen (two successive ejaculates/buck) was collected twice a week via an AV and only ejaculates of >2500 x 10(6) sperm/ml and 70% SPM were diluted in one step at 30 degrees C with the CDE, cooled to 5 degrees C over a 4h-period, frozen in the form of 0.30 ml pellets and stored in liquid nitrogen for 72 h. The results revealed that post-thaw SPM of semen diluted at a rate of 1:4 was significantly (P < 0.01) higher than that of semen diluted at the other rates. Dilution of semen at a rate of 1:19 (< or =151 x 10(6) sperm/ml) not only minimized (P < 0.01) pre-freeze and post-thaw SPM, but also augmented (P < 0.01) pre-freeze and post-thaw rates of lipid peroxidation as evidenced by the high level of MAL production and the ability of antioxidants (1mg/ml EDTA, 200 U/ml bovine liver catalase, 0.61 mg/ml reduced glutathione and 0.11 mg/ml sodium pyruvate) to restore (P < 0.01) pre-freeze and post-thaw SPM. Frozen semen pellets exposed to dry thawing had a greater percentage of SPM (P < 0.01) as well as lower values of SAA and MAL (P < 0.01) than those exposed to wet thawing. Although the kidding rates did not vary significantly among does in natural (55.26%) and synchronized (53.85%) estrus, a higher (P < 0.05) prolificacy was obtained after their insemination in natural (1.81+/-0.16) rather than in synchronized (1.22+/-0.11) estrus.