J Roca

University of Murcia, Murcia, Murcia, Spain

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Publications (187)336.07 Total impact

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    ABSTRACT: This study evaluated two cryoprotectant (CPA) combinations, ethylene-glycol (EG)+dimethyl sulfoxide (DMSO) and EG+propylene glycol (PG), used for the vitrification of germinal vesicle porcine oocytes (GV). In Experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after in vitro maturation (IVM) (18.1 ± 2.3% and 19.4 ± 2.6% for EG+DMSO and EG+PG, respectively) compared with control oocytes (7.6 ± 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In Experiment 2, the percentages of live vitrified-warmed (VW) GV oocytes at 2 hours after warming (EG+DMSO: 67.0 ± 2.3 and EG+PG: 57.6 ± 2.3) were lower than that of fresh control GV oocytes (97.3 ± 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in VW oocytes (EG+DMSO: 59.8 ± 2.3 and EG+PG: 56.2 ± 2.6) than in the control (1.6 ± 1.3). Fertilization efficiency was higher (P < 0.05) in the EG+PG (39.6 ± 2.4) and control (42.0 ± 2.2) groups than the EG+DMSO (26.3 ± 7.7). The cleavage and blastocyst formation rates of the EG+DMSO (25.9 ± 3.5% and 6.6 ± 2.5%, respectively) and EG+PG (20.2 ± 5.4% and 4.7 ± 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 ± 2.7% and 31.9 ± 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG+DMSO was lower. Blastocysts formation was similar for both CPAs combinations.
    Theriogenology 04/2015; DOI:10.1016/j.theriogenology.2015.04.004 · 1.85 Impact Factor
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    ABSTRACT: Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.
    Andrology 01/2015; DOI:10.1111/andr.309 · 3.37 Impact Factor
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    ABSTRACT: This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = −0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = −0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = −0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters. Mol. Reprod. Dev. 2014. © 2014 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 12/2014; 82(1). DOI:10.1002/mrd.22444 · 2.68 Impact Factor
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    ABSTRACT: This study evaluated the effects of mineral oil (MO) overlay during maturation, fertilization, and embryo culture on the timing of nuclear maturation, the progesterone concentrations in the maturation medium, and the subsequent developmental competence of the oocyte. The results from experiment 1 showed that under the typical humidity of laboratory incubators (95%-97%), the culture media osmolality increased in the absence of oil overlay. For this reason, in experiment 2, maturation, fertilization, and embryo culture media were incubated with either an oil cover (MO group) or a microenvironment system for maximum humidity (HM group). Under these conditions, the media osmolality was maintained below 300 mOsm/kg. A portion of oocytes (n = 1414; four replicates) was removed from the maturation medium at 4- to 6-hour intervals to evaluate the nuclear maturation stage. The corresponding medium was used for progesterone measurement. The remaining oocytes were inseminated with frozen-thawed ejaculated sperm and cultured for 12 hours (n = 305) or 7 days (n = 619) to assess fertilization and embryo development parameters, respectively. The progesterone concentration of the maturation medium of the MO group was lower than 1.5 ng/mL at each time point evaluated. The values obtained at 12 hours of maturation and at the end of maturation were 20 and 55 times lower than those of the HM group, respectively. However, compared with the HM group, oil overlay did not delay oocyte progression to metaphase I and II and did not influence normal fertilization, cleavage, blastocyst formation, and total cell number in blastocysts. In conclusion, despite its pronounced impact on progesterone concentration, the use of MO did not affect the time course of oocyte maturation or oocyte developmental competence. Copyright © 2014 Elsevier Inc. All rights reserved.
    Theriogenology 11/2014; 83(4). DOI:10.1016/j.theriogenology.2014.11.001 · 1.85 Impact Factor
  • REPRODUCTION IN DOMESTIC ANIMALS; 10/2014
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    ABSTRACT: The aim of this study was to evaluate the sperm quality in chilled canine semen using different cooling rates from room temperature (23 °C) to 5 °C and subsequently cold stored at 5°C for up to 96 h. In experiment one, semen samples from five dogs were pooled, diluted in Tris-Fructose-Citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5°C using different cooling rates of 2.25, 0.9, 0.45 and 0.2 (control)°C/min. In experiment two, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72 and 96 h of storage at 5°C. The total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using computer-assisted analysis system (CASA) and the percentage of viable spermatozoa was determined using flow cytometry (H-42/PI//FITC-PNA). The cooling rate did not influence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively influenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 h at 5 °C in a Tris-Fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.
    Theriogenology 09/2014; DOI:10.1016/j.theriogenology.2014.05.022 · 1.85 Impact Factor
  • Reproduction Fertility and Development 08/2014; DOI:10.1071/RD14088 · 2.58 Impact Factor
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    ABSTRACT: Excellent fertility and prolificacy have been reported after non-surgical deep uterine transfers of fresh in vivo-derived porcine embryos. Unfortunately, when this technology is used with vitrified embryos, the reproductive performance of recipients is low. For this reason and because the embryos must be stored until they are transferred to the recipient farms, we evaluated the potential application of non-surgical deep uterine transfers with in vivo-derived morulae cultured for 24 h in liquid stage. In Experiment 1, two temperatures (25°C and 37°C) and two media (one fully defined and one semi-defined) were assessed. Morulae cultured in culture medium supplemented with bovine serum albumin and fetal calf serum at 38.5°C in 5% CO2 in air were used as controls. Irrespective of medium, the embryo viability after 24 h of culture was negatively affected (P<0.05) at 25°C but not at 37°C compared with the controls. Embryo development was delayed in all experimental groups compared with the control group (P<0.001). Most of the embryos (95.7%) cultured at 37°C achieved the full or expanded blastocyst stage, and unlike the controls, none of them hatched at the end of culture. In Experiment 2, 785 morulae were cultured in the defined medium at 37°C for 24 h, and the resulting blastocysts were transferred to the recipients (n = 24). Uncultured embryos collected at the blastocyst stage (n = 750) were directly transferred to the recipients and used as controls (n = 25). No differences in farrowing rates (91.7% and 92.0%) or litter sizes (9.0±0.6 and 9.4±0.8) were observed between the groups. This study demonstrated, for the first time, that high reproductive performance can be achieved after non-surgical deep uterine transfers with short-term cultured morulae in a defined medium, which opens new possibilities for the sanitary, safe national and international trade of porcine embryos and the commercial use of embryo transfer in pigs.
    PLoS ONE 08/2014; 9(8):e104696. DOI:10.1371/journal.pone.0104696 · 3.53 Impact Factor
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    ABSTRACT: The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.
    Journal of Reproduction and Development 07/2014; DOI:10.1262/jrd.2014-024 · 1.64 Impact Factor
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    ABSTRACT: This study aimed to evaluate the effect of recipient-donor estrous cycle synchrony on recipient reproductive performance after nonsurgical deep-uterine (NsDU) embryo transfer (ET). The transfers (N=132) were conducted in recipients sows that started estrus 24 h before (-24 h; N=9) or 0 h (synchronous; N=31), 24 h (+24 h; N=74) or 48 h (+48 h; N=18) after the donors. A total of 30 day 5 morulae or day 6 blastocysts (day 0=onset of estrus) were transferred per recipient. The highest farrowing rates (FRs) were achieved when estrus appeared in recipients 24 h later than that in the donors (81.1%), regardless of the embryonic stage used for the transfers. The FR notably decreased (P<0.05) when recipients were -24 h asynchronous (0%), synchronous (61.3%) or +48 h asynchronous (50%) relative to the donors. No differences in litter size (LS) and piglet birth weights were observed among the synchronous and +24 h or +48 h asynchronous groups. While a +24 h asynchronous recipient was suitable for transfers performed with either morulae (FR, 74.3%; LS, 9.2 ± 0.6 piglets) or blastocysts (FR, 84.6%; LS, 9.8 ± 0.6 piglets), a +48 h asynchronous recipient was adequate for blastocysts (FR, 87.5%; LS, 10.4 ± 0.7 piglets) but not for morulae (FR, 30.0%; LS, 7.3 ± 2.3 piglets). In conclusion, this study confirms the effectiveness of the NsDU-ET technology and shows that porcine embryos tolerate better a less advanced uterine environment if they are nonsurgically transferred deep into the uterine horn.
    Journal of Reproduction and Development 07/2014; DOI:10.1262/jrd.2014-022 · 1.64 Impact Factor
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    ABSTRACT: Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
    Cryobiology 07/2014; 69(2). DOI:10.1016/j.cryobiol.2014.07.004 · 1.64 Impact Factor
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    ABSTRACT: BackgroundSex allocation of offspring in mammals is usually considered as a matter of chance, being dependent on whether an X- or a Y-chromosome-bearing spermatozoon reaches the oocyte first. Here we investigated the alternative possibility, namely that the oviducts can recognise X- and Y- spermatozoa, and may thus be able to bias the offspring sex ratio.ResultsBy introducing X- or Y-sperm populations into the two separate oviducts of single female pigs using bilateral laparoscopic insemination we found that the spermatozoa did indeed elicit sex-specific transcriptomic responses. Microarray analysis revealed that 501 were consistently altered (P-value < 0.05) in the oviduct in the presence of Y-chromosome-bearing spermatozoa compared to the presence of X-chromosome-bearing spermatozoa. From these 501 transcripts, 271 transcripts (54.1%) were down-regulated and 230 transcripts (45.9%) were up-regulated when the Y- chromosome-bearing spermatozoa was present in the oviduct. Our data showed that local immune responses specific to each sperm type were elicited within the oviduct. In addition, either type of spermatozoa elicits sex-specific signal transduction signalling by oviductal cells.ConclusionsOur data suggest that the oviduct functions as a biological sensor that screens the spermatozoon, and then responds by modifying the oviductal environment. We hypothesize that there might exist a gender biasing mechanism controlled by the female.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-293) contains supplementary material, which is available to authorized users.
    BMC Genomics 05/2014; 15(1):293. DOI:10.1186/1471-2164-15-293 · 4.04 Impact Factor
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    ABSTRACT: To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome-bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome-bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.
    Theriogenology 05/2014; DOI:10.1016/j.theriogenology.2014.05.008 · 1.85 Impact Factor
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    ABSTRACT: The constitutive 70kDa heat shock protein, HSPA8 has previously been shown to contribute to the long-term survival of spermatozoa inside the mammalian female reproductive tract. Here, we show that a recombinant form of HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances in vitro fertilization performance and decreases sperm mitochondrial activity. Fluorescence recovery after photobleaching (FRAP) revealed that the repair of membrane damage is achieved by an almost instantaneous increase in sperm membrane fluidity. The ability of HSPA8 to influence membrane stability and fluidity, as well as its conserved nature among mammalian species, supports the idea that this protein protects sperm survival through membrane repair mechanisms.
    Reproduction 02/2014; 147(5). DOI:10.1530/REP-13-0631
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    ABSTRACT: 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 × 10(6) 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.
    Journal of Reproduction and Development 02/2014; 60(2). DOI:10.1262/jrd.2013-073 · 1.64 Impact Factor
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    ABSTRACT: To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intra-boar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15-17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in 3 experiments. X- and Y-chromosome-bearing sperm were simultaneously separated according to the Beltsville sperm sorting technology using a high-speed flow cytometer. In the first experiment, inter-boar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome-bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the ROC curve (0.88, p<0.001). In the second experiment, a certain degree of intra-boar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sorteability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent five months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 h of storage at 15-17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine AI programs.
    Theriogenology 01/2014; · 1.85 Impact Factor
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    ABSTRACT: The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10(6) unsorted sperm per oviduct; (2) single LI with 0.5 × 10(6) sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10(6) sex-sorted sperm per oviduct and 0.5 × 10(6) sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10(6) sperm per oviduct and 1 × 10(6) sperm per uterine horn; or (2) 1 × 10(6) sperm per oviduct and 2 × 10(6) sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10(6) sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.
    Theriogenology 10/2013; 81(2). DOI:10.1016/j.theriogenology.2013.09.031 · 1.85 Impact Factor
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    ABSTRACT: The aim of this study was to evaluate the influence of Hoechst 33342 (H-42) concentration and of the male donor on the efficiency of sex-sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10(6) sperm/ml, split into four aliquots, stained with increasing H-42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non-viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X- and Y-chromosome-bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H-42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H-42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex-sorting of canine spermatozoa by flow cytometry can be performed successfully using H-42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.
    Reproduction in Domestic Animals 10/2013; DOI:10.1111/rda.12238 · 1.18 Impact Factor
  • 09/2013; 3(4):40-47. DOI:10.2527/af.2013-0032
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    ABSTRACT: The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008-2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season's influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.
    Cryobiology 09/2013; 67(3). DOI:10.1016/j.cryobiol.2013.09.001 · 1.64 Impact Factor

Publication Stats

3k Citations
336.07 Total Impact Points

Institutions

  • 1991–2015
    • University of Murcia
      • • Department of Animal Medicine and Surgery
      • • Faculty of Veterinary
      Murcia, Murcia, Spain
  • 2001
    • Swedish University of Agricultural Sciences
      Uppsala, Uppsala, Sweden