E. Mocé

Effect of freezing–thawing protocols on the performance of semen from three rabbit lines after artificial insemination

The effect of different freezing and thawing protocols on the results observed after artificial insemination with semen from three different rabbit lines (two maternal lines selected for litter size at weaning, lines A and V, and one line selected for growth rate from weaning to slaughter, line R) was studied. The sperm were frozen with a Tris-citric acid-glucose extender which included 1.75 M DMSO and 0.05 M sucrose as cryoprotectants. The straws were cooled to 5 degrees C for 45 min and then some of them were frozen in a freezer at -30 degrees C for 30 min, whereas the other group of straws were frozen in liquid nitrogen vapor (LNV, 5 cm above the liquid nitrogen level) for 10 min. Straws were thawed at two different temperatures: 50 or 70 degrees C for 10-12s. Significant differences were observed between freezing-thawing protocols, obtaining better results in fertility rate (percentage of pregnant females) when sperm had been frozen in LNV (fertility rate increased between 30 and 50 points in all the lines); the best prolificacy was observed when sperm had been frozen in LNV and thawed at 50 degrees C (70% versus 32% fertility rate, P<0.01 and 7.4 versus 5.9 total number of young born, P<0.01 when sperm had been frozen in LNV or at -30 degrees C and thawed at 50 degrees C, respectively). As for the rabbit line, significant differences were observed between lines in fertility rate (62 and 68% versus 45% fertility rate for lines A, V and R, P<0.01), and total number of young born (5.8 versus 6.9 versus 4.6 total number of young born for lines A, V and R, P=0.02). The best results for all lines in both fertility and total number of young born were observed when sperm had been frozen in LNV and thawed at 50 degrees C (85% versus 84% versus 50% fertility rate and 6.7 versus 8.3 versus 7.3 total number of young born for lines A, V and R, respectively), when compared to the results of the control group, frozen at -30 degrees C and thawed at 50 degrees C (30% versus 52% versus 19% fertility rate and 6.7 versus 6.4 versus 4.5 total number of young born for lines A, V and R, respectively). In conclusion, the best results (fertility rate and prolificacy) for all the rabbit lines were obtained after freezing in liquid nitrogen vapor and thawing at 50 degrees C, being more pronounced in the line selected for high growth rate (line R).

Effect of an asynchrony between ovulation and insemination on the results obtained after insemination with fresh or frozen sperm in rabbits

The effects of the introduction of an 8-h asynchrony between ovulation and insemination on litter size components from rabbits were assessed. A total of 202 females belonging to a maternal line were used. Fresh and frozen sperm were used to perform the inseminations. Sperm was frozen with an extender composed of 1.75 M DMSO and 0.05 M sucrose. Four experimental groups were obtained depending on the type of sperm used (fresh or frozen) and on the moment that ovulation had been induced relative to the insemination (at the same time as insemination (t(0)) or 8 h before insemination (t(8))). Laparoscopy was performed on 12th day of pregnancy in pregnant females, and the ovulation rate, normal and total implanted embryos were noted. At kindling, total and live-born rabbits were noted. Results showed that better results were obtained after insemination with fresh semen than with frozen sperm (for females in the group t(0): 79% versus 61% fertility rate, 10.2 versus 6.4 normal implanted embryos and 8.1 versus 5.2 total number born, for fresh and frozen sperm, respectively). On the other hand, after the introduction of an 8-h asynchrony between ovulation and insemination, results were lower for both fresh (50% fertility rate, 7.5 normal implanted embryos and 5.7 total number born for the group of the asynchrony) and frozen sperm (31% fertility rate, 4.6 normal implanted embryos and 3.4 total number born for the group of the asynchrony). Although an approach between the moment of insemination and ovulation is justified when sperm survival could be compromised, results observed after the induction of an 8-h asynchrony were not those expected, perhaps due to the ageing of the oocytes before being fertilised, leading to both lack of fertilisation or early embryonic mortality.

Efficiency of Repeated In Vivo Oocyte and Embryo Recovery After rhFSH Treatment in Rabbits

This study aims to assess the efficiency of in vivo oocyte and embryo recovery after a recombinant human FSH (rhFSH) treatment in rabbit does. Females were distributed in two experimental groups: donor does were treated with rhFSH (superovulation group) for 3 days prior to artificial insemination (embryo recovery) or ovulation induction (oocyte recovery) and does without treatment remained as the control group. Mature oocytes or embryos were collected with the laparoscopy technique 16 h after ovulation induction (oocytes) or 72 h after artificial insemination (embryos). Up to four recoveries were performed with each doe. Recovery efficiencies differed significantly between embryos (84%) and oocytes (58%). Yet, the recovery rates for the superovulation and control groups did not differ. The rhFSH group was associated with a significant increase (p < 0.05) in the number of oocytes and embryos recovered in comparison with the control group (10.2 +/- 1.0 and 14.3 +/- 1.2 vs 6.0 +/- 2.7 and 8.4 +/- 2.3 for oocytes and embryos, respectively). Results from this study indicate that repeated in vivo oocyte and embryo recovery from rhFSH superovulated does maximizes the number of oocytes or embryos collected from the same female.

Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 degrees C, 5% CO(2), and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH+10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH+5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14+/-0.074 and 0.15+/-0.076 vs. 0.46+/-0.078 and 0.50+/-0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.

Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate

Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses.

Effect of Cooling Rate to 5°C, Straw Size and Farm on Fertilizing Ability of Cryopreserved Rabbit Sperm

High fertility and prolificacy in rabbits are currently only achieved using fresh sperm. This study was conducted to determine if the cooling rate to 5 °C, the straw size and the farm where artificial inseminations are performed have an impact on the fertilizing ability of rabbit sperm cryopreserved with an extender containing dimethyl sulphoxide (DMSO; 1.75 m) and sucrose (0.05 m). Slow cooling to 5 °C improved neither fertility rate (58 vs 56% kindling rate for fast and slow cooling, respectively) nor prolificacy (6.5 vs 8.7 total born for slow and fast cooling, respectively; p < 0.05) compared to fast cooling rate to 5 °C. The straw size did not have an effect on either fertility or prolificacy (47 vs 57% kindling rate and 6.3 vs 6.8 total born for sperm loaded into 0.25 and 0.5 ml straws, respectively). In addition, similar results were obtained between farms (46-57% kindling rate and 4.9-6.7 total born), although this effect should be studied further. In conclusion, with this extender, slow cooling does not present a beneficial effect on sperm fertilizing ability and either 0.25 or 0.5 ml straws can be used to freeze the sperm, obtaining similar results after artificial insemination. In addition, similar results were obtained between farms when using cryopreserved sperm, and these results were lower than those obtained after artificial insemination with fresh semen. Therefore, new approaches are needed to improve the results obtained when cryopreserved sperms are used before this type of sperm can be used for commercial purposes.

Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (°Cmin(-1)) from -6°C to -100°C using a programmable freezer: slow (-15°Cmin(-1), S), medium (-40°Cmin(-1), M) or fast (-60°Cmin(-1), F). After thawing (50°C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P≤0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15°Cmin(-1)) and fast (-60°Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability.

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P<0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P<0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability.

Freezability genetics in rabbit semen

The aim of this study was to estimate the heritability of semen freezability and to estimate the genetic correlation between frozen-thawed sperm traits and the growth rate in a paternal rabbit line. Estimated heritabilities showed that frozen-thawed semen traits are heritable (ranged between 0.08 and 0.15). In the case of Live-FT (percentage of viable sperm after freezing), the estimated heritability is the highest one, and suggests the possibility of effective selection. After the study of genetic correlations it seems that daily weight gain (DG) was negatively correlated with sperm freezability, but no further conclusions could be drawn due to the high HPD95%. More data should be included in order to obtain better accuracy for the estimates of these genetic correlations. If the results obtained at present study were confirmed, it would imply that selection for DG could alter sperm cell membranes or seminal plasma composition, both components related to sperm cryoresistance.