Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile.

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma.

BACKGROUND: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). METHODS: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90ß was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. RESULTS: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90ß differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90ß-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). CONCLUSIONS: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

Seminal Extracellular Vesicles and Their Involvement in Male (In)Fertility: A Systematic Review.

Seminal plasma contains numerous extracellular vesicles (sEVs). Since sEVs are apparently involved in male (in)fertility, this systematic review focused on studies specifically investigating such relationship. Embase, PubMed, and Scopus databases were searched up to 31 December 2022, primarily identifying a total of 1440 articles. After processing for screening and eligibility, 305 studies were selected as they focused on sEVs, and 42 of them were considered eligible because they included the word fertility or a related word such as infertility, subfertility, fertilization, and recurrent pregnancy loss in the title, objective(s), and/or keywords. Only nine of them met the inclusion criteria, namely (a) conducting experiments aimed at associating sEVs with fertility concerns and (b) isolating and adequately characterizing sEVs. Six studies were conducted on humans, two on laboratory animals, and one on livestock. The studies highlighted some sEV molecules, specifically proteins and small non-coding RNAs, that showed differences between fertile and subfertile or infertile males. The content of sEVs was also related to sperm fertilizing capacity, embryo development, and implantation. Bioinformatic analysis revealed that several of the highlighted sEV fertility-related proteins would be cross-linked to each other and involved in biological pathways related to (i) EV release and loading and (ii) plasma membrane organization.

Extracellular vesicles would be involved in the release and delivery of seminal TGF-ß isoforms in pigs.

Introduction: Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor ß (TGF-ß), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-ßs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-ßs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-ß isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-ß isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-ß isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-ß isoforms and in their safe transport from the male to the female reproductive tract.

Immunological uterine response to pig embryos before and during implantation.

The establishment of a successful pregnancy can only occur through a concerted functioning of the entire female reproductive system, allowing for fertilization, subsequent embryo development and implantation of the conceptus. In this context, the uterine immunological responses responsible for rejection or tolerance of the conceptus are of critical importance. The aim of the present review is to summarize our current knowledge about those cellular and molecular immunological events occurring at the uterine level during pre-implantation and implantation stages of pregnancy in the pig. Advancing our understanding of the immune mechanisms involved in the success or failure of pregnancy will provide cues to develop novel strategies augmenting endometrial receptivity, finally increasing the efficiency of assisted reproductive technologies in pigs.

Extracellular vesicles in seminal fluid and effects on male reproduction. An overview in farm animals and pets.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles released by most functional cells to body fluids, containing bioactive molecules, mainly proteins, lipids, and nucleic acids having actions at target cells. The EVs have essential functions in cell-to-cell communication by regulating different biological processes in target cells. Fluids from the male reproductive tract, including seminal plasma, contain many extracellular vesicles (sEVs), which have been evaluated to a lesser extent than those of other body fluids, particularly in farm animals and pets. Results from the few studies that have been conducted indicated epithelial cells of the testis, epididymis, ampulla of ductus deferens and many accessory sex glands release sEVs mainly via apocrine mechanisms. The sEVs are morphologically heterogeneous and bind to functional cells of the male reproductive tract, spermatozoa, and cells of the functional tissues of the female reproductive tract after mating or insemination. The sEVs encapsulate proteins and miRNAs that modulate sperm functions and male fertility. The sEVs, therefore, could be important as reproductive biomarkers in breeding sires. Many of the current findings regarding sEV functions, however, need experimental confirmation. Further studies are particularly needed to characterize both membranes and contents of sEVs, as well as the interaction between sEVs and target cells (spermatozoa and functional cells of the internal female reproductive tract). A priority for conducting these studies is development of methods that can be standardized and that are scalable, cost-effective and time-saving for isolation of different subtypes of EVs present in the entire population of sEVs.

Intrauterine Infusion of TGF-ß1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development.

Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor ß1 (TGF-ß1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-ß1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. On day 6 (day 0-onset of estrus), all "donors" were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant "donors" and cyclic "recipients," incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-ß1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

Delays in processing and storage of pig seminal plasma alters levels of contained antioxidants.

Seminal plasma (SP) antioxidants are considered biomarkers of sperm function and fertility for AI-boars. The current protocol for their measurement implies the SP was harvested immediately after ejaculation and prompt stored at -80 °C until analysis. Such protocol may be impractical for AI-centers. This study evaluated how SP levels of antioxidants were influenced by delays in (1) SP-harvesting (0 [control], 2 or 24 h at 17 °C after ejaculate collection), in (2) SP-freezing (0 [control] or 24 h at 17 °C after SP-harvesting) or (3) the temperature of storage (-80 °C [control] or - 20 °C). The SP-antioxidants evaluated were: glutathione peroxidase [GPx], superoxide dismutase [SOD], paraoxonase-1 [PON-1], trolox equivalent antioxidant capacity [TEAC] and oxidative stress index [OSI]. A total of 120 aliquots from 10 entire ejaculates were handled in three trials. They were centrifuged (1500 g, 10 min) for harvesting SP and antioxidants were measured with an Automatic Chemistry Analyzer. A 24 h-delay in harvesting the SP led to an increase (p˂0.001) in TEAC and SOD SP-levels, and a decrease (p˂0.05) of OSI and PON-1. Similarly, a 24 h-delay to freeze the SP increased (p˂0.01) TEAC values and decreased (p˂0.01) PON-1 and GPx activity levels. Finally, storing the SP at -20 °C decreased (p˂0.001) SP-levels of TEAC, PON-1 and GPx, and increased (p˂0.01) OSI values. Strong positive relationships (p˂0.001) were found between antioxidant SP-levels in processed samples and their respective controls. In sum, handling and SP storage influence antioxidant measurements in AI-boars. Reliable levels of SP-antioxidants can only be warranted if a strict protocol for harvesting and SP storage is followed.

Blastocyst-Bearing Sows Display a Dominant Anti-Inflammatory Cytokine Profile Compared to Cyclic Sows at Day 6 of the Cycle.

In the context of porcine embryo transfer (ET) technology, understanding the tightly regulated local uterine immune environment is crucial to achieve an adequate interaction between the transferred embryos and the receiving endometrium. However, information is limited on the uterine immune status of cyclic-recipient sows when receiving embryos during ET. The present study postulated that the anti- and proinflammatory cytokine profile 6 days after the onset of estrus differs between endometria from uninseminated cyclic sows and blastocyst-bearing sows. On Day 6 of the cycle, endometrial explants were collected from sows inseminated or not inseminated during the postweaning estrus and cultured for 22 h. The culture medium was then analyzed for the contents of a total of 16 cytokines using Luminex MAP® technology. The results showed important differences in the endometrial production of most cytokines between the sow categories, with a predominant anti-inflammatory environment displayed by the blastocyst-bearing endometria. These findings suggest that sperm, seminal plasma (SP) and/or early embryos modify the uterine environment by inducing an immune-tolerant cytokine profile already visible at Day 6. Whether the SP or some of its active components may help to develop strategies to maximize the reproductive performance of recipients after ET needs further investigation.

Measurable Cytokine Concentrations in Pig Seminal Plasma Are Modified by Semen Handling and Storage.

Sample handling and storing are critical steps for the reliable measurement of circulating biomolecules in biological fluids. This study evaluates how cytokine measurements in pig seminal plasma (SP) vary depending on semen handling and SP storage. Thirteen cytokines (GM-CSF, IFNγ, IL-1α, IL-1ß, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNFα) were measured using Luminex xMAP® technology in individual seminal plasma (SP) samples (n = 62) from healthy breeding boars. Three separate experiments explored the delay (2 h and 24 h) in SP collection after ejaculation (Experiment 1) and SP storage, either short-term (5 °C, -20 °C and -80 °C for 72 h, Experiment 2) or long-term (at -20 °C and -80 °C for two months, Experiment 3), before analysis. Levels in fresh SP-samples were used as baseline control values. Delays in SP harvesting of up to 24 h did not substantially impact SP cytokine measurements. Some cytokines showed instability in stored SP samples, mainly in long-term storage. Ideally, cytokines in pig SP should be measured in fresh samples harvested within 24 h after ejaculation. If storage of SP is imperative, storage conditions should be adjusted for each cytokine.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is fully expressed in the genital tract, seminal plasma and spermatozoa of male pigs.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine identified in boar seminal plasma (SP) but until now unexplored in terms of place of production and its association to spermatozoa. This study aimed to explore these aspects by evaluating the presence of GM-CSF in porcine reproductive organs (testes, epididymis and accessory sex glands), SP and mature spermatozoa (from cauda epididymis and ejaculated) using Western blot (WB), immunohistochemistry and immunocytochemistry. Positive labelling was obtained in tissues, SP and spermatozoa. In reproductive organs, WB revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa). In SP and epididymal fluid, the GM-CSF appeared only in its active form while in spermatozoa the GM-CSF form present varied among sperm sources. Non-viable spermatozoa showed more GM-CSF than viable spermatozoa (14.87 ± 1.98 RU vs. 7.25 ± 0.52 RU) of fluorescence intensity. In conclusion, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to SP. Consequently, the GM-CSF ought to regulate male genital tract and sperm function as well as mediating initial inflammatory responses and further mediating later immune actions by the female to semen deposition.

Seminal Plasma Modulates miRNA Expression by Sow Genital Tract Lining Explants.

The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.

Period of Boar Ejaculate Collection Contributes to the Yearly Intra-Male Variability of Seminal Plasma Cytokines.

The concentrations of cytokines in seminal plasma (SP) fluctuate over time in healthy males, weakening their practical usefulness as diagnostic tools. This study evaluated the relevance of intra-male variability in SP cytokines and to what extent the period of the year when ejaculate is collected contributes to such variability. Thirteen cytokines (GM-CSF, IFNγ, IL-1α, IL-1ß, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and TNFα) were measured using the Luminex xMAP® technology for 180 SP samples of ejaculate collected over a year from nine healthy and fertile boars. The SP samples were grouped into two annual periods according to decreasing or increasing daylight and ambient temperature. Intra-male variability was higher than inter-male variability for all cytokines. All SP cytokines showed concentration differences between the two periods of the year, showing the highest concentration during the increasing daylength/temperature period, irrespective of the male. Similarly, some cytokines showed differences between daylength/temperature periods when focusing on their total amount in the ejaculate. No strong relationship (explaining more than 50% of the total variance) was found between annual fluctuations in SP-cytokine levels and semen parameters. In conclusion, the period of the year during which ejaculates were collected helps explain the intra-male variability of SP-cytokine levels in breeding boars.

Achievements and future perspectives of embryo transfer technology in pigs.

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.

Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos.

The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation (IVM) or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 µM CoQ10. The addition of 10-50 µM CoQ10 to the IVM medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2-4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 µM) in the maturation medium negatively affected blastocyst rates. In conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.

Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs.

Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract. Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos. Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation. Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others. Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.

Comparison between ultrasonographic findings of benign and malignant canine mammary gland tumours using B-mode, colour Doppler, power Doppler and spectral Doppler.

The aim of this study was to evaluate whether the comparison between the ultrasonographic features of canine mammary tumours, assessed by B-Mode, colour Doppler, power Doppler, spectral Doppler, and histopathologic features, would help to differentiate if a tumour is benign or malignant. Ultrasonographic examinations of 104 tumours were performed. Volume, margins, presence of a capsule, echotexture and presence and distribution of the vascular flow of the tumours were evaluated. All the tumours were surgically removed, submitted for histopathologic examination and classified in two groups: Group I (benign tumours) and Group II (malignant tumours). Echotexture was the only parameter evaluated by B-Mode ultrasonography where significant differences were found (p<0.01), with tumours in Group I being homogeneous and tumours in Group II presenting greater heterogeneity. Presence of vascular flow was observed in most of the tumours from both groups and no differences between them were found. Regarding flow distribution, significant differences were observed between groups (p<0.05). In benign tumours, the most common vascular pattern was the peripheral, showing significant differences (p<0.05) compared to mixed and central patterns. In malignant tumours the mixed pattern was the most frequent. Also significant differences among other patterns (peripheral and central) were found. Concerning vascular resistivity and pulsatility indexes, there were no significant differences between the two groups. The echotexture and type of vascular flow pattern of canine mammary gland tumours may help, in a first examination of the tumour, to differentiate between benign and malignant tumours; however to reach a definitive diagnosis histological study is required.

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.

The effects of atracurium on bispectral index (BIS) values in dogs anaesthetized with isoflurane.

Balanced anaesthesia techniques employ specific drugs in addition to general anaesthetics. The potential effect of some of these drugs on the bispectral index (BIS) remains unclear. BIS seems to be unaffected by the administration of neuromuscular blockers while opioids may block nociceptive-induced BIS increases in anaesthetized humans. In this study, the effect of a single dose of atracurium on BIS values was assessed in dogs premedicated with morphine and anaesthetized with isoflurane (1 MAC). Fifteen female dogs undergoing elective ovariohysterectomy were employed. BIS values were recorded before and after the administration of atracurium. These values were then recorded again once the animals were surgically stimulated. The end tidal isoflurane concentration, cardiovascular and respiratory parameters were recorded through the study. Results show that the mean (±SD) BIS values decreased slightly, but significantly (P=0.033) from 67±8.42 to 65±5.84 after the administration of atracurium. Despite significant increase in heart rate and arterial pressure during the surgical phase, the mean BIS values were not modified by ovariohysterectomy. Atracurium appeared to have minimal clinical effect on the BIS in anaesthetized dogs.

Pentoxifylline added to freezing or post-thaw extenders does not improve the survival or in vitro fertilising capacity of boar spermatozoa.

This study evaluated whether pentoxifylline added to freezing and thawing extenders influenced the function of boar spermatozoa. In Experiment 1, pooled ejaculated sperm-rich fractions were frozen in 0.5 ml straws after dilution in extender supplemented with pentoxifylline to a final concentration of 0, 2, 4, 8, 16 or 32 mM. The addition of 4, 8, 16 and 32 mM pentoxifylline to the freezing extender significantly decreased the progressive and total motility of spermatozoa. The percentage of viable spermatozoa with intact acrosomes as well as the penetration rate and the efficiency of fertilisation were significantly lower in pentoxifylline-treated groups compared with the untreated control. In Experiment 2, a pool of three straws with 'good' post-thaw sperm quality parameters and another three straws with 'poor' sperm quality were diluted in extender with 0, 1, 2, 4, 8, 16 or 32 mM pentoxifylline. Post-thaw samples with both 'good' and 'poor' sperm quality with 0, 2, 4, 8 and 16 mM were used to assess IVF parameters. The addition of pentoxifylline to post-thaw extender did not improve the post-thaw motility or viability of spermatozoa compared with the control. The in vitro penetration was higher (P<0.05) than the control for oocytes fertilised with spermatozoa that were thawed and incubated in extender with 4, 8 and 16 mM pentoxifylline. However, no differences were observed in the efficiency of fertilisation. We conclude that pentoxifylline, as a supplement added to the freezing extender, has a deleterious effect and that it does not improve the survival or in vitro fertilising efficiency of frozen-thawed boar spermatozoa when added after thawing.