The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa.

BACKGROUND: Sex 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. RESULTS: By 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. CONCLUSIONS: Our 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.

Effects of rapid cooling prior to freezing on the quality of canine cryopreserved spermatozoa.

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.

Effects of Hoechst 33342 staining and ultraviolet irradiation on mitochondrial distribution and DNA copy number in porcine oocytes and preimplantation embryos.

Hoechst 33342 (H342), in combination with ultraviolet (UV) irradiation, is frequently used to aid or confirm the enucleation of porcine oocytes in somatic cell nuclear transfer programs. The exposure of oocytes to H342 and UV irradiation has a deleterious effect on the development of in vitro-fertilized porcine oocytes, with increasing exposure to UV irradiation (up to 30 sec) having more drastic effects. It has been hypothesized that this decrease in embryonic development could be due to damage to the mitochondrial DNA (mtDNA). To investigate this hypothesis, we analyzed the mitochondrial distribution and DNA copy number of in vitro-matured porcine oocytes exposed to H342/UV and the subsequent embryonic development compared with the mitochondrial distribution and DNA copy number of in vivo-derived oocytes and embryos. Using quantitative, real-time polymerase chain reaction (qPCR) protocols to analyze mtDNA and confocal laser scanning microscopy with MitoTracker Deep Red to determine mitochondrial distribution, we demonstrated that the simultaneous exposure of in vitro-matured porcine oocytes to H342 staining and UV irradiation is associated with reduced oocyte developmental competence and abnormal mitochondrial distribution in the resulting cleaved embryos. In addition, 2- to 4-cell embryos derived from oocytes exposed to H342/UV showed a significant decrease in mtDNA copy number. These results should be considered when H342/UV procedure is used during nuclear transfer in recipient porcine oocytes.

Boar seminal plasma: current insights on its potential role for assisted reproductive technologies in swine.

Seminal plasma (SP) supports not only sperm function but also the ability of spermatozoa to withstand biotechnological procedures as artificial insemination, freezing or sex sorting. Moreover, evidence has been provided that SP contains identifiable molecules which can act as fertility biomarkers, and even improve the output of assisted reproductive technologies by acting as modulators of endometrial and embryonic changes of gene expression, thus affecting embryo development and fertility beyond the sperm horizon. In this overview, we discuss current knowledge of the composition of SP, mainly proteins and cytokines, and their influence on semen basic procedures, such as liquid storage or cryopreservation. The role of SP as modulator of endometrial and embryonic molecular changes that lead to successful pregnancy will also be discussed.

Influence of insemination time on the fertility of sex sorted frozen-thawed Y-sperm in red deer.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon® PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01-55:30 h; I_2, 55:31-56:00 h; I_3, 56:01-56:30 h; or, I_4, 56:31-57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples.

Optimization of protocols for Iberian red deer (Cervus elaphus hispanicus) sperm handling before sex sorting by flow cytometry.

Currently, sperm reproductive biotechnologies such as sex sorting and cryopreservation are undoubtedly valuable tools for improving the economic and biological efficiency of red deer production systems. In this context, and because of the particular characteristics of this species (extensive exploitation typically far from laboratory facilities), a key goal is to optimize the design of an adequate handling protocol of sperm samples before samples are subjected to sex sorting and cryopreservation procedures to obtain better outputs from the application of these technologies. The main aim of this paper was to design an adequate protocol for Iberian red deer sperm handling before sex sorting by flow cytometry to obtain optimal yields when sex sorting is used in this species. Semen samples from 11 adult males were obtained by electroejaculation during the breeding season. In this study, we tested different protocols for the handling of Iberian red deer spermatozoa before sorting by using different concentrations of sperm (400 or 800 × 106) and adding or not Hoechst 33342 before the transport of samples to the sorting facilities. Based on the results, the most adequate method used to handle samples before sorting was transportation at a high sperm concentration (800 × 106/mL) without Hoechst 33342. These transportation conditions in combination with Hoechst 33342 staining at 5.2 µL/mL once at the flow cytometry laboratory resulted in better (P < 0.05) sorting efficiency (99.9% of the samples showing split) than both, those samples transported at 400 × 106sperm/mL (between 51.2 and 55.2% of the samples showing split) and those samples stained before transport at a sperm concentration of 400 × 106sperm/mL (between 15.4 and 75.7% of the samples showing split). Sorting rates and sperm quality after sorting and cryopreservation was not affected (P > 0.05) by sperm handling before sorting. Moreover, the sorting yields were compatible with the practical application of these reproductive biotechnologies.

Improving the efficiency of sperm technologies in pigs: the value of deep intrauterine insemination.

The use of AI in pigs has dramatically expanded in the last few years. New methodological advances in AI are required to serve the requirements of new sperm technologies, such as the use of low dose AI, because the use of cervical AI has a very low efficiency leading to low fertility results. One of the strategies devised to meet these requirements is the deposition of semen near the site of fertilization in the oviduct. Using deep intrauterine insemination with a specially designed catheter, a 20-fold reduction in the number of freshly and diluted inseminated spermatozoa can be achieved without decreasing farrowing rates. Moreover, an advantage of deep intrauterine insemination is the possibility of using processed, 'weaker' spermatozoa such as those that have been frozen-thawed or sex-sorted. Although deep intrauterine insemination should be of benefit to the pig industry, more investigations are needed to understand the mechanisms related to sperm colonization of the oviducts and identify the minimal sperm numbers needed to obtain maximal fertility results for processed and unprocessed boar spermatozoa.

Effects of centrifugation before freezing on boar sperm cryosurvival.

Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. Sperm recovery (Bürker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P <.05) values were achieved using 2400 x g for 5 or 3 minutes and 1600 x g for 5 minutes, which showed no differences (P >.05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Centrifugation regimes C1 and C2 showed significantly (P <.05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P <.05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P >.05) by the centrifugation regime used. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol.

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa.

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.

Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar spermatozoa.

The present study was carried out to determine the pregnancy rates, farrowing rates and litter size in sows with either induced or spontaneous ovulation inseminated with flow cytometric sorted spermatozoa using deep intrauterine insemination technology. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry/cell sorting but not separated into separate X and Y populations. In Experiment 1, sows (n=200) were weaned and treated for estrus/ovulation induction with eCG/hCG. Inseminations with either sorted (70 or 140 million) or non-sorted (70 or 140 million) spermatozoa were done using a specially designed flexible catheter. Farrowing rates were 39.1 and 78.7% for 70 million of sorted and non-sorted, respectively, and 46.6 and 85.7% for 140 million of sorted and non-sorted, respectively (P<0.05). The litter size in sows inseminated with sorted spermatozoa showed a tendency to be lower than when non-sorted spermatozoa were inseminated. In Experiment 2, sows (n=140) were inseminated as in Experiment 1 except that natural estrus was used. The ovaries of these sows were evaluated by transrectal ultrasonography. Farrowing rates were 25 and 77.2% for 70 million of sorted and non-sorted, respectively, and 32 and 80.9% for 140 million of sorted and non-sorted, respectively (P<0.05). These results show that the Deep Intrauterine Insemination technology can be successfully used to produce piglets from sorted spermatozoa when sows are hormonally treated to induce synchronous post weaning oestrus and ovulation.