Modelling bodyweight to avoid anthelmintic underdosing of goats in resources-limited settings.

Improved management of livestock in resource-limited settings can provide a means towards improved human nutrition and livelihoods. However, gastrointestinal nematodes (GIN) are a significant production-limiting factor. Anthelmintics play a role in GIN management; however, few anthelmintic classes are available in many low-middle-income countries. Utilising a limited range of classes may increase selection for anthelmintic resistance; therefore, strategies to reduce other selective pressures are of heightened importance. Avoiding anthelmintic underdosing is one such strategy, but it can be challenging without access to accurate bodyweight measurement. Many previous studies have used thoracic girth as a practical proxy for bodyweight in goats; however, they have rarely considered the potential impact of natural variation on therapeutic doses. Here, the relationship between bodyweight and thoracic girth was modelled using data from 820 goats from three Malawian biomes in two seasons, with the specific aim of avoiding underestimation of bodyweight. The internally cross-validated linear regression (∛Weight ~ 0.053 + 0.040*Girth, R2 = 0.92, rounded up to the nearest 5 kg) was validated against data from an additional 352 Malawian goats (1.4% of goats allocated an underdose and 10.2% allocated a dose > 200% of bodyweight). The equation was further externally validated using an historical dataset of 150 goats from Assam, India (2.7% of goats were allocated to an underdose and 24.8% allocated to a > 200% of bodyweight). These results suggest that a more globally generalisable approach may be feasible, provided the accuracy of the estimate is considered alongside the therapeutic index of the pharmaceutical.

Conjunctival mucous membrane colour as an indicator for the targeted selective treatment of haemonchosis and of the general health status of peri-urban smallholder goats in southern Malawi.

The world's growing population is becoming increasingly centred around large cities, affording opportunities for peri-urban food production. Goats are well-suited to conversion of resources that are available in peri-urban settings into meat and occasionally milk. Haemonchus contortus has been described as "the nemesis of small ruminant production systems in tropical and subtropical regions"; hence control of haemonchosis through planned animal health management affords a pragmatic first step in improving the production efficiency of peri-urban goats. This study of peri-urban goat production investigated the potential value of targeted selective treatment of haemonchosis. 452 peri-urban goat keepers in southern Malawi were visited during three seasonal periods with relevance to the epidemiology of haemonchosis. 622, 599 and 455 individually identified goats were clinically examined during the dry season, the rainy season, and shortly after the end of the rainy season, respectively. Data were recorded for sex, age, weight, conjunctival mucous membrane colour score (FAMACHA©), body condition score (BCS) and faecal worm egg count (FEC); and where possible for pregnancy and lactation status. Animals with pale ocular mucous membranes were treated with 10 mg/kg albendazole, then re-examined 14 days later. Animals with pink mucous membranes, but FECs ≥250 eggs per gram were also re-examined and treated 14 days later. The results show high variability in growth rates deduced from the ages and bodyweights of each of 999 goats at the time of their enrolment. FAMACHA© scores alone were a poor index for the targeted selective treatment of haemonchosis, because they failed to identify too many animals that would have required treatment at different times of year and using different FAMACHA© and FEC cut-offs. Combining the indices of FAMACHA© scores ≥4, body condition scores ≥2, and age >18 months was more reliable in identifying those animals requiring treatment when different epidemiologically-relevant FEC thresholds for different seasons were taken into account. Inclusion of late pregnancy or early lactation status would have resulted in very few animals requiring treatment being missed. The use of conjunctival mucous membrane colour scoring in this way provided a valuable insight of the general health status of the peri-urban goats, to create opportunities for planned animal health management to improve productivity. The efficacy of albendazole treatment was poor, putatively due to drug resistance, or poor drug bioavailability in goats. In summary, our study shows opportunities for better production efficiency in peri-urban goats, and demonstrates the value of simple clinical diagnostic indices as decision support tools in planned animal health management.

Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure.

Persistent organic pollutants (POPs) can induce epigenetic changes in the paternal germline. Here, we report that folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs in a rat model. Pregnant founder dams were exposed to an environmentally relevant POPs mixture (or corn oil) ± FA supplementation and subsequent F1-F4 male descendants were not exposed to POPs and were fed the FA control diet. Sperm miRNA profiles of intergenerational (F1, F2) and transgenerational (F3, F4) lineages were investigated using miRNA deep sequencing. Across the F1-F4 generations, sperm miRNA profiles were less perturbed with POPs+FA compared to sperm from descendants of dams treated with POPs alone. POPs exposure consistently led to alteration of three sperm miRNAs across two generations, and similarly one sperm miRNA due to POPs+FA; which was in common with one POPs intergenerationally altered sperm miRNA. The sperm miRNAs that were affected by POPs alone are known to target genes involved in mammary gland and embryonic organ development in F1, sex differentiation and reproductive system development in F2 and cognition and brain development in F3. When the POPs treatment was combined with FA supplementation, however, these same miRNA-targeted gene pathways were perturbed to a lesser extend and only in F1 sperm. These findings suggest that FA partially mitigates the effect of POPs on paternally derived miRNA in a intergenerational manner.

Implementation of high coverage mass rabies vaccination in rural Uganda using predominantly static point methodology.

Despite successful eradication programmes in many regions, rabies remains responsible for approximately 60,000 human deaths annually, and no country in Africa is rabies-free. Dogs are the principal reservoir of the virus in Africa and the World Health Organisation recommends that at least 70% of the dog population be vaccinated in order to break the transmission cycle. Most attempts at mass rabies vaccinations in Africa have failed to vaccinate high numbers of dogs at a high coverage. Successful studies have often used a door-to-door (DTD) approach, which is logistically challenging and expensive compared to a static point (SP) approach. Mission Rabies has successfully implemented a combined SP and DTD method in cities in India and Malawi. This campaign used a combined methodology in rural Uganda, starting with a SP campaign, followed by a DTD campaign, and then subsequent transect surveys to assess vaccination coverage. This was facilitated by the use of a smartphone application which recorded all vaccinations and survey responses along with their Global Positioning System location. A total of 4172 dogs were vaccinated in 7 days, attaining an estimated 88.4% coverage. This campaign is of particular note as 95.9% of the vaccinations were performed at SPs. The human-to-dog ratio was 4.9 with a mean dogs per house of 1.2. Most dogs were owned (93.7%). This demonstrates that high-number, high-coverage vaccination is achievable in rural Uganda and provides data that may refine future campaign approaches.

Prenatal exposure to an environmentally relevant mixture of Canadian Arctic contaminants decreases male reproductive function in an aging rat model.

Elevated levels of organochlorines (OC) have been reported in Inuit populations in the Arctic. We hypothesized that prenatal exposure to a Canadian Arctic OC mixture adversely affects male reproductive function and health with age. Sprague-Dawley female rats (F0) were gavaged with an environmentally relevant concentration of an Arctic OC mixture or corn oil (Control) during mating with untreated males until parturition (F1 litters). After postnatal day (PND) 90, the weights of the OC F1 males differed dramatically relative to Controls (P<0.05; n=10) and they exhibited respiratory distress. Except for possible thinning of the alveolar barrier, histological observation of the lungs revealed no apparent pathology to explain the respiratory distress. At PND 365, OC F1 males had reduced relative reproductive organ weights and lower sperm quality than Controls (P<0.05). At PND 90, OC F1 males were subfertile (P<0.05), but were infertile at PND 365. In conclusion, environmentally relevant prenatal OC exposure reduced reproductive function and health in aging male rats, providing new insight into the effects of early-life exposures to these contaminants.

Preservation of Domesticated Honey Bee (Hymenoptera: Apidae) Drone Semen.

Preservation of honey bee (Apis mellifera L., Hymenoptera: Apidae) sperm, coupled with instrumental insemination, is an effective strategy to protect the species and their genetic diversity. Our overall objective is to develop a method of drone semen preservation; therefore, two experiments were conducted. Hypothesis 1 was that cryopreservation (-196 °C) of drone semen is more effective for long-term storage than at 16 °C. Our results show that after 1 yr of storage, frozen sperm viability was higher than at 16 °C, showing that cryopreservation is necessary to conserve semen. However, the cryoprotectant used for drone sperm freezing, dimethyl sulfoxide (DMSO), can harm the queen and reduce fertility after instrumental insemination. Hypothesis 2 was that centrifugation of cryopreserved semen to reduce DMSO prior to insemination optimize sperm quality. Our results indicate that centrifuging cryopreserved sperm to remove cryoprotectant does not affect queen survival, spermathecal sperm count, or sperm viability. Although these data do not indicate that centrifugation of frozen-thawed sperm improves queen health and fertility after instrumental insemination, we demonstrate that cryopreservation is achievable, and it is better for long-term sperm storage than above-freezing temperatures for duration of close to a year.

Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

Review article: dermatological complications of immunosuppressive and anti-TNF therapy in inflammatory bowel disease.

BACKGROUND: With the expanding list of medications available to treat patients with inflammatory bowel disease (IBD), it is important to recognise adverse events, including those involving the skin. Dermatological adverse events may be confused with extra-intestinal manifestations of IBD. AIM: To review drug-related dermatological manifestations associated with immunosuppressive and anti-tumour necrosis factor (anti-TNF) therapy. METHODS: The literature was searched on PubMed for dermatological adverse events in IBD. RESULTS: Present thiopurine exposure was associated with a 5.9-fold [95% confidence interval (CI), 2.1-16.4] increased risk of developing non-melanoma skin cancer (NMSC). The peak incidence is highest in Caucasians over the age of 65 years with crude incidence rates of 4.0 and 5.7/1000 patient-years for present and previous use. In anti-TNF-exposed subjects, drug-induced lupus was reported in 1% of the cases and a psoriatic rash in up to 3% of the cases. Anti-TNF monotherapy increases the risk of NMSC ~2-fold to a rate of 0.5 cases per 1000 person-years. Cutaneous lymphomas have been rarely reported in subjects on thiopurine or anti-TNF drug monotherapy. Combination therapy seems to have an additive effect on the risk of developing NMSC and lymphoma. CONCLUSIONS: Physicians need to be aware of the wide spectrum of dermatological complications of immunosuppressive and anti-TNF therapy in IBD, especially psoriasis and non-melanoma skin cancer. Vigilance and regular screening for non-melanoma skin cancer is recommended. Case discussions between gastroenterologists and dermatologists should be undertaken to best manage dermatological adverse events.

Expression of neuronal markers in the endometrium of women with and those without endometriosis.

STUDY QUESTION: How do the expression patterns of neuronal markers differ in the endometrium of women with and without endometriosis? SUMMARY ANSWER: The neuronal markers, PGP9.5, NGFp75 and VR1, are expressed in the endometrium at levels that do not differ between women with and without endometriosis. WHAT IS KNOWN ALREADY: Aberrant neuronal growth within the uterus may contribute to abnormal fertility and uterine dysfunction. However, controversy still exists as to whether aberrant innervation in the endometrium is associated with gynaecological pathology such as endometriosis. This may reflect the use of subjective methods such as histology to assess the innervation of the endometrium. We, therefore, employed a quantitative method, western blotting, to study markers of endometrial innervation in the presence and absence of endometriosis. STUDY DESIGN, SIZE, DURATION: This study included 45 women undergoing laparoscopic examination for the diagnosis of endometriosis. Endometrial samples were analysed by western blot for the expression of neuronal and neurotrophic markers, PGP9.5, VR1 and NGFp75. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Endometrial pipelle biopsies were obtained from patients with (n = 20, study group) and without (n = 25, control group) endometriosis. Tissue was analysed by immunohistochemistry and western blot analysis for the expression of pan-neuronal marker, PGP9.5, sensory nociceptive marker, TPVR1, and low-affinity neurotrophic growth factor receptor, NGFRp75. MAIN RESULTS AND THE ROLE OF CHANCE: PGP9.5, NGFp75 and VR1 were expressed in the endometrium of women, independent of the presence of endometriosis. Furthermore, the expression level of PGP9.5, VR1 and NGFp75 did not alter between the two cohorts of women. LIMITATIONS, REASONS FOR CAUTION: Studies of this nature are subject to the heterogeneous nature of patient population and tissue samples despite attempts to standardize these parameters. Hence, further studies using similar methodology will be required to confirm our results. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight that sensory neuronal markers are present in women with and without endometriosis. Future work will assess what the targets of the endometrial nerves are and investigate their function, their impact on endometrial biology and, in particular, whether aberrant neuronal function, rather than the mere presence of neuronal function, could be the root cause of subfertility and/or pain affecting many endometriosis sufferers. Our results do not, however, confirm the previous paradigm of increased innervation in the endometrium of women with endometriosis, nor the use of nerve cell detection from pipelle biopsies to diagnose endometriosis.

Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices.

Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications.

Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved.

To evaluate the influence of dietary supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) on storage of boar semen, three experiments were conducted: two involved long-term, fresh semen storage (Exp. 1 and Exp. 2), whereas the other involved cryopreservation (Exp. 3). Boars were allocated randomly to three dietary treatments (for 6-7 mo). In addition to a daily allowance of 2.5 kg of a basal diet, they received: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO), containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA); or 3) 60 g of tuna oil (TO), containing 33% DHA and 6.5% EPA. In Experiment 1 (n = 26) and Experiment 2 (n = 18), semen was cooled and stored in vitro for several days at 17 °C before assessment, whereas in Experiment 3 (n = 18), viability, motility, acrosomal integrity, susceptibility to peroxidation (LPO), and DNA fragmentation were determined in fresh and frozen-thawed sperm. In Experiment 1, sperm from boars fed TO had better resistance to fresh storage; even after 7 or 9 d of storage at 17 °C, there were more (P = 0.03) motile sperm in boars fed TO (>60%) than in those fed AF or MO. In Experiment 2, fish oil supplementation did not influence any aspect of sperm quality during semen storage (P > 0.10). In Experiment 3, cryopreservation decreased the proportion of motile and viable frozen-thawed sperm as well as acrosomal integrity and increased DNA fragmentation and LPO (P < 0.01) relative to fresh semen, although sperm quality was unaffected by treatments (P > 0.09). In conclusion, although adding fish oil to the diet failed to significantly improve the quality of cryopreserved boar sperm, inconsistent responses of long-term storage of cooled sperm to dietary n-3 PUFA supplementation warrant further investigation.

Effect of dietary n-3 fatty acids (fish oils) on boar reproduction and semen quality.

The aim of this study was to evaluate the effects of dietary supplementation with different fish oils (rich in PUFA) vs. hydrogenated animal fat (SFA) on semen production and quality, fatty acid composition, and preservation properties in boars under controlled and commercial conditions. In Exp. 1 (in a research station), 44 boars, allocated to 4 dietary treatments, received daily 2.5 kg of basal diet with a supplement of 1) 62 g of hydrogenated animal fat (AF, n = 12); 2) 60 g of menhaden oil containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA; MO, n = 11); 3) 60 g of tuna oil containing 33% DHA and 6.5% EPA (TO, n = 11); and 4) 60 g of menhaden oil and 2 mg/kg of biotin (MO+B, n = 10). Biotin is a critical factor in the elongation of PUFA. Semen was collected according to 3 successive phases: phase 1 (twice per week for 4 wk); phase 2 (daily collection for 2 wk); and phase 3 (twice per week for 10 wk). Experiment 2 was conducted in commercial conditions; 222 boars were randomly allocated to AF, MO, and TO treatments. Semen was collected twice weekly over a 6-mo period. All diets were balanced to be iso-energetic and provided an equivalent of 989 mg of vitamin E per day. Classical measurements of sperm quantity and quality were done for both experiments. Experiment 1 showed, after 28 wk of supplementation, a massive transfer of n-3 PUFA into sperm from boars fed fish oil diets (MO and TO). No differences were observed among dietary treatments for libido (P > 0.30), sperm production (P > 0.20), or percentage of motile cell (P > 0.20). Unexpectedly, MO+B diet reduced the percentage of normal sperm compared with the other treatments (P < 0.03). In conclusion, although it modified the fatty acid composition of sperm, supplementation of boars with dietary fish oils, rich in long chain n-3 fatty acids, did not influence semen production or quality postejaculation.

Effects of dietary vitamin supplementation and semen collection frequency on reproductive performance and semen quality in boars.

The present study was undertaken to assess the relevance of increasing the daily provision of dietary vitamins on vitamin metabolic status and semen characteristics of boars under controlled and commercial conditions as well as to evaluate the efficiency of this vitamin supplement to allow boars to cope with intensive semen collection frequency. In the first experiment, 39 boars were allocated to 2 dietary treatments, a basal diet (control) and the basal diet supplemented with extra fat- and water-soluble vitamins (Vit). Within each treatment, boars were submitted to 2 regimens of semen collection frequency: 3 times per 2 wk (3/2) and 3 times per week (3/1) over a 12-wk period. Afterwards, all boars were intensively collected (daily) for 2 wk. A resting period of 4 wk followed, and all boars were collected 2 times per week. Thereafter, collection frequencies were reversed, and the same procedure was followed until the end of the intensive collection period. A second experiment was conducted in commercial conditions at a commercial stud, and 252 boars were randomly allocated to the control and Vit dietary treatments. All boars were collected 2 times per week over a 6-mo period. Classical measurements of ejaculate and sperm quality were assessed, and blood samples were collected throughout both experiments to quantify vitamin concentrations. In the first experiment, vitamin concentrations in blood and seminal plasma increased in Vit boars (P < 0.05); however, vitamin concentrations were not affected by collection frequency (P > 0.14). The Vit supplement did not affect sperm production or sperm quality (P > 0.28), although semen volume increased during the 12-wk periods for Vit boars (P < 0.05). The 3/1 boars produced fewer doses per ejaculate than 3/2 boars (P < 0.01); however, the cumulative sperm production for the 12-wk periods increased by 19% in 3/1 boars compared with 3/2 boars. In the second experiment, blood plasma concentrations of vitamin B(9) were greater (P < 0.01) in Vit than control boars. The vitamin supplement did not increase sperm production of boars (P > 0.61). In conclusion, dietary supplements of fat- and water-soluble vitamins increase the amount of vitamins available for the animal, and the collection frequencies had no effect on vitamin status. Moreover, in spite of an effect on the ejaculate volume, the dietary supplement of extra vitamins had no effect on sperm production or quality.

Effects of dietary vitamin supplementation and semen collection frequency on hormonal profile during ejaculation in the boar.

To evaluate the effect of dietary and management factors on boar hormonal status during ejaculation, 39 boars were canulated to determine the profiles of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17beta-estradiol (E2), and testosterone (T) in blood plasma and seminal fluid. Prior to canulation, 18 boars were fed a basal diet (control), whereas the remainder (n=21) were fed a basal diet supplemented with extra vitamins (supplemented). Within each dietary treatment, two regimens of semen collection were used over the 3mo preceding the hormonal evaluation: three times per 2wk (3/2) or three times per wk (3/1). Plasma E2 was lower (P<0.01) before ejaculation (232.5+/-22.6pg/mL) than at the onset of ejaculation (255.2+/-27.1ng/mL). Plasma T increased from 5.14+/-0.72, before ejaculation to 5.87+/-0.86ng/mL at the onset of ejaculation in supplemented boars, whereas it decreased from 5.15+/-0.65 to 4.87+/-0.70ng/mL in controls (diet by time, P<0.05). At the onset of ejaculation, plasma FSH was higher in 3/2 boars (0.436+/-0.06ng/mL) than in 3/1 boars (0.266+/-0.04ng/mL; P<0.05). During ejaculation, plasma LH increased linearly (P<0.01) from 0.59+/-0.07 to 0.97+/-0.10ng/mL, and plasma E2 and T concentrations were correlated (r=0.62, P<0.01). Plasma FSH before and during ejaculation was negatively correlated with sperm production (r=-0.60, P<0.01) and testicular weight (r=-0.50, P<0.01). In conclusion, dietary and management factors had few impacts on hormonal profiles during ejaculation, but homeostasis of some hormones was related to some criteria of reproductive performance in boars.

Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa.

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.

Cryopreservation in different concentrations of glycerol alters boar sperm and their membranes.

To test the hypothesis that glycerol would concomitantly affect sperm membrane structure and the function of the intact cells, boar semen (4 ejaculates from 4 boars) was cryopreserved in an egg yolk extender with 0%, 2%, 4%, or 8% glycerol in 0.5-mL straws using previously derived optimal cooling and thawing rates. Increasing glycerol concentrations increased spermatozoal progressive motility immediately after thawing and after 2 hours at 43 degrees C, but decreased the percentage of sperm with normal acrosomal morphology. The mathematical products of the motility and acrosomal integrity scores (MOT x NAR index) were low in 0% and 8% glycerol, and significantly higher in 2% and 4% glycerol. The fluidity of sperm-head plasma membranes, a measure of molecular interaction, was assessed with the lipid probes trans-parinaric acid and cisparinaric acid (tPNA, cPNA), during a 2.5-hour incubation with or without 1 mM Ca2+. Membrane fluidity detected by each probe differed significantly, indicating the presence of at least 2 domains whose constituent molecules had unique dynamics. Behavior of each domain was radically altered by cryopreservation. Increasing glycerol concentration caused a variably faster loss of fluidity in the cPNA domain, and had highly variable effects on fluidity change over time in the tPNA domain. Normal acrosomal ridge (NAR) and the MOT x NAR index correlated significantly with the fluidity of the more mobile cPNA domain (+/- 1 mM Ca2+), supporting the hypothesis of an interrelationship of glycerol concentration during cryopreservation with sperm membrane structure and cell function. The MOT x NAR index may be a useful guide in choosing optimal cryoprotectant concentrations.

Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins.

Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An M(r) 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An M(r) 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.

Impaired maturation, fertilization, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture.

The reproductive health risks related to exposure to persistent organic pollutants in the environment remain controversial. This debate is partly because most studies have investigated only one or two chemicals at a time, whereas populations are exposed to a large spectrum of persistent chemicals in their environment. Using the pig as a toxicological model, we hypothesized that exposing immature cumulus-oocyte complexes to an organochlorine mixture during in vitro maturation (IVM) would adversely affect oocyte maturation, fertilization, and subsequent embryo development. This organochlorine mixture mimics that which contaminates the Arctic marine food chain. Cumulus-oocyte complexes were cultured in IVM medium containing increasing concentrations of the organochlorine mixture, similar to that found in women of highly exposed populations. Organochlorines reduced the quality of cumulus expansion and the viability of cumulus cells in a dose-response manner. The proportion of apoptotic cumulus cells also increased due to organochlorine exposure. Half of the oocytes were fixed after insemination, and the remainders were cultured for 8 days. Concentrations of organochlorines did not affect the rates of oocyte degeneration, sperm penetration, and development to morula. However, incidence of incompletely matured oocytes increased and polyspermy rate decreased, both in a dose-response manner with increasing organochlorine concentrations. Blastocyst formation and number of cells per blastocyst declined with organochlorine concentration. Exposing porcine cumulus-oocyte complexes to an environmentally pertinent organochlorine mixture during IVM disturbs oocyte development, supporting recent concerns that such pollutants harm reproductive health in humans and other mammalian species.