C. Tomás

Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

Treating sperm with cholesterol-loaded cyclodextrins (CLC) improves the cryosurvival of the sperm of different cold-shock sensitive species. However, the response of boar sperm to this treatment is not fully understood. The aim of this study was to determine how CLC and methyl-β-cyclodextrin (MβCD, not loaded with cholesterol) affect the parameters for boar sperm functionality, including sperm osmotic resistance, and the ability of the sperm to capacitate and to penetrate the sows immature oocytes in vitro. Samples treated with CLC or MβCD prior to freezing exhibited similar percentages of motile sperm, live sperm and sperm with intact acrosomes as the control samples (P>0.05). In addition, these treatments did not alter the response of the boar sperm to capacitating conditions. However, when compared to the controls and the MβCD-treated samples, the CLC-treated sperm maintained greater percentages of motile sperm and live sperm in a wide range of osmotic solutions including hypo- (50, 75 and 150 mOsm/kg) and hyper-osmotic (600, 800 mOsm/kg) conditions (P<0.05). In addition, the CLC-treated sperm exhibited greater oocyte penetration ability than the control and the MβCD-treated sperm (P<0.0001). In conclusion, the pre-freezing treatment of boar sperm with CLC does not alter the ability of the sperm to respond to capacitating conditions. Despite not increasing the cryosurvival of the sperm, this treatment widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability.

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.

Is Sperm Freezability Related to the Post-Thaw Lipid Peroxidation and the Formation of Reactive Oxygen Species in Boars?

The aim of the present study was to determine whether the levels of reactive oxygen species (ROS) substances production and the levels of lipid peroxidation of the sperm membrane were related to the quality that the ejaculates exhibited after cryopreservation in boars. Ejaculates from 42 healthy boars were used in this study and they were cryopreserved with the lactose-egg yolk extender (LEY). Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: the percentage of sperm with intact plasma membrane (SIPM), intracellular reactive oxygen substances production through mean of DCF fluorescence intensity of total sperm (mean-DCF) and the percentage of viable and non-viable sperm containing oxidized BODIPY (VSOB and NVSOB). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. The classification of the ejaculates into good or bad freezers was performed through hierarchical cluster analysis from SIPM and TMS at 150 min post-thawing. The ejaculates of those males classified as good freezers exhibited higher (p < 0.05) SPIM, TMS and PMS than the bad freezers, although both groups presented similar (p > 0.05) VSOB, NVSOB and mean-DCF. Therefore, these results show that lipid peroxidation and the amount of reactive oxygen substances in the sperm after cryopreservation are similar between boars classified as good or bad freezers.

In vivo fertilising ability of frozen-thawed boar sperm treated with cholesterol-loaded cyclodextrins prior to cryopreservation

The use of frozen-thawed (FT) sperm for the artificial insemination of pigs is rare. Treating boar sperm with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances the penetration of immature oocytes in vitro, and this phenomenon has been positively correlated with in vivo fertilisation ability in pigs. The objective of this study was to compare the in vivo fertilising ability of boar sperm treated with 0 (control) or 1mg CLC/120×10(6) sperm (CLC) prior to freezing. The fertilising ability of the FT sperm was compared in hormonally treated (equine/human chorionic gonadotropin hormones; eCG/hCG) weaned sows inseminated once (cervical insemination) at the following fixed-times after hCG administration: 37h (experiment 1) or 30h (experiment 2). In experiment 1, both treatments exhibited similar fertility rates of 67.7 and 55.9% for the control and CLC, respectively (P>0.05); however, the CLC group had a smaller litter size (11.3±0.9) than the control group (13.6±0.8) (P<0.05). In experiment 2, the pregnancy and farrowing rates were 65.2 and 66.7% for the control and CLC groups, respectively, and the litter size was 12.9±1 and 11.3±1 for the control and CLC groups, respectively, which were similar (P>0.05) for both treatments. These results indicate that the timing of insemination developed for the FT control sperm may not be suitable for CLC-treated sperm and that CLC-treated sperm may benefit from a shorter time interval between the hCG treatment and insemination. Moreover, acceptable results can be obtained with FT boar sperm with a single artificial insemination performed 30h after hCG treatment, which is the time interval recommended for fresh sperm.

Inclusion of seminal plasma in sperm cryopreservation of Iberian pig

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process.

Effect of the holding time at 15 °C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8h), thawing rate (37 °C for 20s or 70 °C for 8s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing-thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4h post-thawing.

Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin.

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 106 sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P <0.05), even in semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P <0.05). Thus, CLC-treated sperm showed a higher tolerance to high glycerol concentrations (5%) in terms of sperm viability (59.19% ± 3) than non-treated sperm (45.58% ± 3; P<0.05). Therefore, it could be necessary to modify the freezing extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.

Efecto de la inmunocastración en el peso canal y las piezas nobles en cerdas Ibéricas de cebo

A total of 192 Iberian females (Duroc*Iberian), with 110 days of age and 45 ± 3.41 kg BW, were used to study the effect of immunocastration in yield of carcass and prime lean cuts. There were three treatments: surgically castrated females (CC), entire females (CE), and inmunocastrated females (CI) the experimental design was a complete randomized blocks with 48 replicates total (4 pigs in each), 12 replicates per block and 16 per treatment. The feed, the same for all animals, was suministered ad libitum. After sacrifice, 31 pigs were selected for each treatment to assess the performance of carcass and main lean cuts. Treatment CI tended to improve slaughter and carcass weights (p 0.05) and significantly less in the CC (p 55 %; p 0.05) and quadratic for CE and CI (p<0.05). The immunocastration favors the growth of Iberian female; the surgical castration is the least interesting option.