L. Gil

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.

Antioxidant effect of rosemary (Rosmarinus officinalis) on boar epididymal spermatozoa during cryopreservation

The objective of the present study was to evaluate the ability of rosemary to protect epididymal boar spermatozoa from freeze-thaw damage. Testis from eight boars were collected at the slaughterhouse in two trials. In the laboratory, sperm from epididymis were recovered by flushing and cryopreserved in lactose-egg yolk solution supplemented with various concentrations (low; medium; high) of rosemary. After thawing, total motility, viability, acrosome integrity, response to hypoosmotic swelling test (HOST) and malonaldehide (MDA) concentration were assessed. The results showed that there was an increase in motility at 1, 2 and 3 h in the presence of rosemary. The addition of this herb provided a significant beneficial effect on viability at 2 h of incubation, compared to the control group. Conversely, acrosome status was not affected by any extender. Higher concentration of rosemary produced significant improvement in percentages of positive HOST at 0 and 1 h, whereas no impact was observed at the end of incubation. Considering membrane lipid peroxidation, a greater decrease in MDA production was observed when rosemary content was raised. Rosemary-enriched freezing extender improved the post-thaw epididymis boar spermatozoa quality, showing a significant correlation between rosemary concentration and concentration of MDA. Further studies are needed to define the active component in rosemary that prevents peroxidation.

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3h. Results showed that total motility at 1 and 3h, progressive motility at 3h, positive hypoosmotic response at 2 and 3h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.

Dimethylformamide is not better than glycerol for cryopreservation of boar semen.

To improve the boar sperm cryopreservation process, the influence of the sugar (lactose, trehalose) source and the cryoprotectant [glycerol, dimethylformamide (DMF)] on the success of freezing was investigated. Sperm samples were frozen in one of six extenders: lactose plus 3% glycerol (LG); lactose plus 1.5% glycerol and 1.5% DMF (LGD); lactose plus 3% DMF (LD); trehalose plus 3% glycerol (TG); trehalose plus 1.5% glycerol and 1.5% DMF (TGD); trehalose plus 3% DMF (TD). Effects on motility, viability, acrosome integrity and hypoosmotic test (HOST) were measured. The results showed that extender containing 3% glycerol retained the highest motility percentages. In regard to viability and acrosome integrity, all extenders yielded similar rates except for the decreasing values of TD. Endosmosis was diminished in TD and LD at 2 h (P = 0.0018), as compared with the others. The results of the study demonstrated that the use of DMF as a cryoprotectant adversely affected boar sperm quality after cryopreservation.

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 (0 μM, 26.25 μM, 52.5 μM and 105 μM). 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 120 min post-thawing. The plasma and acrosomal membrane integrity were improved by supplementation with 105 μMRA (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 240 min post-thawing, the samples supplemented with 105 μMRA showed the lowest DNA oxidation rate (P<0.05). The penetration rate was significantly higher on spermatozoa cryopreserved with 105 μMRA (P<0.05). The results suggest that RA provides a protection for boar spermatozoa against oxidative stress during cryopreservation by their antioxidant properties.

Cryopreservation of epididymal stallion sperm.

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallions breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.

Fennel (Foeniculum vulgare) provides antioxidant protection for boar semen cryopreservation

Boar semen is extremely vulnerable to cold shock and it is also sensitive to peroxidation due to the high content of unsaturated fatty acids in the plasma membrane. Antioxidants exert a protective effect on the plasma membrane of frozen boar sperm. Fennel has been shown to contain antioxidant substances. Therefore, this study was performed to evaluate the effect of different concentrations of fennel added to the freezing extender on boar semen quality and lipid peroxidation after thawing. Semen collected from four boars was cryopreserved in lactose‐egg‐yolk extender or in the same extender with varying concentration of fennel essences: low (LF); medium (MF); high (HF). Analysis of data clearly indicated that higher concentrations of fennel produced significant improvement in total motility. Moreover, when fennel was included in the extender, a dose‐dependent tendency to increase sperm viability was observed. In contrast, the addition of fennel had no effect on acrosome integrity or hypoosmotic swelling test (HOST) compared with the control. Malondialdehyde (MDA) formation decreased significantly in fennel groups, yielding similar results for MF and HF. Fennel seems a new antioxidant for use in sperm cryopreservation, but its particular effects on sperm physiology must be further studied, especially the causes of motility stimulation and its effect on lipoxidation.

Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96® extender.

The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservation in combination with different cryoprotectants: Acetal (5%), Dimethylformamide (5%) and Glycerol (5%), alone and combined (2.5% each) on ejaculated and epididymal spermatozoa. Ejaculates collected from mature stallion and epididymal sperm samples were cryopreserved in INRA® varying content of cryoprotectant and cryopreserved. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. We conclude that INRA 96® is suited as extender for freezing when it is used in combination with Dimethylformamide (5%) or Dimethylformamide (2.5%)+Glycerol (2.5%) for samples of ejaculate. The combination of Dimethylformamide (2.5%)+Glycerol (2.5%) showed the best results on epididymal spermatozoa. In conclusion, the combination of Dimethylformamide and Glycerol as cryoprotectants in INRA® medium enhanced equine epididymal and ejaculated spermatozoa quality after cryopreservation.

Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen.

Cryopreservation of boar semen is still considered suboptimal due to the low fertility when compared with fresh semen. This study was performed to evaluate the effects of green tea (Camellia sinensis) supplementation of the freezing extender at different concentration (0, 2.5%, 5%, 10%) and also to determine the influence of increasing holding time from 2 to 24 h at 15 °C. Seventeen ejaculates from nine boars were used to make pools of three of them and then cryopreserved. Sperm motility, viability, acrosome integrity, membrane functionality (HOST) and capacitation status were determined before freezing and at 0, 30, 60, 90 and 120 min after thawing. Lipid peroxidation was evaluated just after thawing. The main findings emerging from this study were the following: (i) no improvement in quality of thawed spermatozoa with addition of tea to the freezing extender, (ii) no improvement in quality of thawed spermatozoa with prolonged holding time, (iii) lower peroxidation rate in presence of tea 5% and (iv) a decrease in the number of uncapacited viable spermatozoa with any tea supplementation. We conclude that amplification of holding time in semen cryopreservation process does not vary results, facilitating freezing protocol. Tea supplementation reduces lipoxidation but did not improve quality parameters.

Current status of freeze-drying technology to preserve domestic animals sperm.

In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at room temperature or 4°C, thereby resulting in enormous reductions in storage and shipping costs. Unlike sperm cryopreserved after gradual freezing, the sperm membrane may be further damaged by both snap-freezing and drying stresses during the FD procedure. As mammalian spermatozoa lose their motility, viability and, at least partially, their DNA integrity when freeze-dried, they must be microinjected into an oocyte by intracytoplasmic sperm injection (ICSI). Although the efficiency of ICSI is limited when freeze-dried spermatozoa are used, embryos and live offspring can be produced. DNA fragmentation in freeze-dried spermatozoa is one of the main causes of failure of embryonic development and successful pregnancy. In this regard, it has been suggested that endonucleases are among the leading causes of DNA fragmentation in spermatozoa along with oxidative stress caused by the release of reactive oxygen species (ROS). Many factors influence the FD process, and it is not clear how FD affects specific components of sperm from different animal species. As such, a sound understanding of the FD process would result in increased production of embryos and/or live offspring. The aim of this review was to study the various stages and techniques used in the FD process and to further evaluate the results obtained.