Ylva Sjunnesson

Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva

BackgroundThe purpose of this study was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) developed for measuring free cortisol in human saliva and total cortisol concentration in diluted human serum, for its applicability in measuring cortisol concentration in pig saliva. Collection of saliva is less stressful than e.g. blood sampling, and is a non-invasive method.FindingsSaliva was collected by allowing sows to chew on cotton swabs held by forceps. Thereafter, the swabs were centrifuged to retrieve the saliva. The ELISA was performed according to instructions provided by the manufacturer. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV was below 10% and inter-assay CV below 15% for samples of high, medium and low cortisol concentrations. The mean recovery was 117% and the linearity and parallelism showed an r2-value of 0.994 and 0.993, respectively. For biological assessment of induced social stress, two saliva samples were collected in the morning from 6 primiparous and 21 multiparous sows. One sample was collected when the sows were individually housed in a farrowing pen and a second sample was collected when the sows were group housed. The primiparous sows had a significant higher cortisol concentration compared to the multiparous sows when group housed.ConclusionThe results obtained in this validation study indicate that the ELISA is suitable for measuring cortisol concentration in porcine saliva.

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

BackgroundGood quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility.MethodsThe semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 103, 8 x 103 and 24 x 103 spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis.ResultsTotal and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment.ConclusionThe use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted.

Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential.

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL-1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

The tolerance of feline corpus and cauda spermatozoa to cryostress.

Epididymal sperm preservation can be used to avoid the total loss of genetic material in threatened species. Spermatozoa from the corpus, as from the cauda, are motile and can undergo capacitation. Thus, they can potentially be preserved for assisted reproductive technologies. However, cryopreservation of spermatozoa has a direct detrimental effect on sperm quality. The aim of this study was to compare the chromatin stability and the survival rate of spermatozoa from the corpus and cauda epididymis after cryopreservation. Epididymal spermatozoa were collected and cryopreserved from the corpus and cauda of 12 domestic cats. Sperm motility, progressive motility, membrane integrity, acrosome integrity, and DNA integrity were evaluated before and after freezing thawing. The average total number of spermatozoa collected from the corpus was lower (10.2 × 10(6) ± 7.4) than that from the cauda epididymis (24.9 × 10(6) ± 14.4; P = 0.005). The percentage of spermatozoa with intact DNA did not differ significantly whether it was collected from the corpus or cauda regions and did not decrease after freezing thawing in either region. However, motility of spermatozoa from both regions was affected by the freezing thawing process with a significant decline in motility after thaw compared with fresh spermatozoa. A significant difference in the percentage of motile sperm between the corpus and cauda was observed after the freezing thawing process (P < 0.001). Although sperm motility was lower in postthaw spermatozoa from the corpus epididymidis than from the cauda, the rate of the reduction did not differ between regions. This study indicates that the cryopreservation process does not have a negative effect on chromatin stability of feline epididymal spermatozoa. Spermatozoa from the corpus region have a similar freezability as spermatozoa from the cauda region. Therefore, preservation of spermatozoa from the corpus and the cauda epididymidis might be of value in preserving genetic material from endangered or valuable felids.

The ability of feline spermatozoa in different epididymal regions to undergo capacitation and acrosome reaction

The sperm maturation process that occurs in the epididymis is a necessary process for spermatozoa to acquire motility and the ability to undergo capacitation, which is an important key for fertilization. The aim of this study was to evaluate the ability of feline spermatozoa from different regions of the epididymis to undergo capacitation and acrosome reaction. Experiment I: epididymal spermatozoa from caput, corpus and cauda regions were placed in phosphate buffered saline (control medium) and in vitro fertilization medium (capacitating conditions). Sperm motility, motility patterns, plasma membrane integrity and tyrosine phosphorylation were evaluated at time 0 and 60min after incubation. Experiment II: spermatozoa were treated with 2μM of calcium ionophore (A23187) to induce the acrosome reaction and acrosome reaction was evaluated. The results showed a significant effect of region with a higher percentage of tyrosine phosphorylation in spermatozoa from the cauda than in the caput or corpus regions (P=0.0061; P=0.0088). Spermatozoa from corpus and cauda showed higher values in the majority of the measured motility parameters than spermatozoa from the caput (P<0.0001). Spermatozoa from all epididymal regions can undergo the acrosome reaction in vitro in response to induction by calcium ionophore with no difference between regions (P>0.05). Spermatozoa from all epididymal regions were able to undergo capacitation. Higher percentage of tyrosine phosphorylation in spermatozoa from the cauda reflect that they more easily underwent capacitation compared to spermatozoa from caput and corpus which required more time of incubation for capacitation. In conclusion feline epididymal spermatozoa from all regions can undergo capacitation and acrosome reaction in vitro and do not require incubation under capacitating conditions.

Hyperinsulinemia during in vitro oocyte maturation changes gene expression of insulin signaling in bovine Day-8 embryos

The obesity-metabolic-syndrome-complex is a growing problem in humans as in other species and known to be associated with decreased fertility. Insulin concentrations differ from physiological levels during periods of metabolic imbalance. The dairy cow suffers from metabolic disturbances due to a stressed metabolism caused by high milk-production.

Practical applications of sperm selection techniques for improving reproductive efficiency

Several selection techniques are available for processing spermatozoa. Apart from sperm washing to remove seminal plasma, only “swim-up” and colloid centrifugation have been used to any extent to prepare spermatozoa for in vitro fertilization, and only colloid centrifugation has been used to prepare sperm samples for artificial insemination. Single-layer centrifugation (SLC) through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility, normal morphology and intact chromatin in a range of species. This method is less timeconsuming than swim-up, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The applications of SLC are as follows: to improve sperm quality in insemination doses or in samples for in vitro fertilization, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.

Consequences for Piglet Performance of Group Housing Lactating Sows at One, Two, or Three Weeks Post-Farrowing.

Housing lactating sows with piglets in a multi-suckling pen from around 14 days post-farrowing is common practice in Swedish organic piglet production. However, nursing-suckling interaction is less frequent in multi-suckling pens than in individual farrowing pens, thus affecting piglet performance, e.g., piglet growth. Moreover, piglet mortality is higher in systems using multi-suckling pens. Three management routines whereby lactating sows with piglets were moved from individual farrowing pens to multi-suckling pens at one, two, or three weeks post-farrowing were compared in terms of nursing-suckling interaction and piglet performance. Correlations between nursing-suckling interaction, piglet performance, and piglet mortality were also examined. In total, 43 Yorkshire sows with piglets were included in the study. Nursing-suckling interaction and all piglet performance parameters except piglet mortality did not differ between management routines. Piglet mortality in the individual farrowing pens did not differ between management routines, but piglet mortality in the multi-suckling pen was lower (P<0.05) when piglets were group housed at three weeks compared with one week post-farrowing. Overall piglet mortality was positively correlated with mortality in the multi-suckling pen for piglets group housed at one week (r = 0.61: P<0.05) and at two weeks post-farrowing (r = 0.62: P<0.05) but not for piglets group housed at three weeks post-farrowing. In conclusion, overall piglet mortality could be reduced if sows and piglets are group housed at three weeks post-farrowing and piglet survival the first week post-farrowing is improved.

A comparison study of insulin concentrations in follicular fluid, serum and in vitro-production of bovine embryos – risks of generating unfavourable metabolic conditions during early development

Insulin has frequently been used as a stimulatory factor in routine in vitro embryo production (IVP) and is added in supra-physiological concentrations to different media. Meanwhile, insulin as a key metabolic hormone is elevated in patients with metabolic syndrome or diabetes, syndromes known to impair fertility.

Lipid profile of bovine blastocysts exposed to insulin during in vitro oocyte maturation

Insulin is a key hormone with important functions in energy metabolism and is involved in the regulation of reproduction. Hyperinsulinaemia is known to impair fertility (for example, in obese mothers); therefore, we aimed to investigate the impact of elevated insulin concentrations during the sensitive period of oocyte maturation on gene expression and lipid profiles of the bovine Day-8 embryo. Two different insulin concentrations were used during in vitro oocyte maturation (INS10=10µgmL-1 and INS0.1=0.1µgmL-1) in order to observe possible dose-dependent effects or thresholds for hyperinsulinaemia in vitro. By investigating gene expression patterns by an mRNA microarray in combination with lipid profile analysis by desorption electrospray ionisation-mass spectrometry (DESI-MS) of embryos derived from insulin-treated oocytes, we gained further insights regarding molecular responses of embryos to insulin provocation during the first days of development. Lipid metabolism appeared to be influenced on multiple levels according to gene expression results but the profiles collected in positive-ion mode by DESI-MS (showing mostly ubiquinone, cholesteryl esters and triacylglycerols) did not differ significantly from controls. There are parallels in follicular development of ruminants and humans that make this bovine model relevant for comparative research on early human embryonic development during hyperinsulinaemia.