Bacteriospermia among smallholder artificial insemination boars in the Philippines and potential associated factors

Objective:To determine the prevalence of bacteriospermia,the bacterial load,and the potential factors associated with bacterial contamination in boar semen collected by local smallholder artificial insemination operators.Methods:Fifteen individual raw semen samples were collected from locally available artificial insemination boars owned by different smallholder boar operators within the 5th district of Leyte,Philippines and were subjected to standard bacteriological culture and identification,including a survey of potentially associated factors.Prevalence and bacterial count were determined accordingly,while boar characteristics and collection practices were clustered following agglomerative hierarchical clustering technique.Results:One hundred percent contamination with a bacterial count of(2.01±0.38)×10^(3) CFU/mL was observed.At least 73.33%of the samples were positive for Bacillus spp.,while other identified isolates included Enterobacter spp.,Staphylococcus spp.,E.coli,Pseudomonas spp.,Citrobacter spp.,and Klebsiella spp.Conclusions:Despite the high prevalence of bacteriospermia,the bacterial count is low.Nevertheless,on-farm practices on boar health and management,semen collection,and sanitation as well as the enhancement of basic protocols to control contamination should be conscientiously considered in smallholder artificial insemination operation.

Serologic and molecular survey for major viral pathogens in grazing hybrid wild boars in Northeast China

Hybrid wild boar husbandry is an important component of livestock production in Northeast China.However,the current disease situation of these animals is largely unknown due to a lack of disease surveillance.The present study was conducted to determine the prevalence of several important viral diseases in the hybrid wild boar population of Northeast China.Between September 2015 to December 2016,169 blood and 61 tissue samples were collected from apparently healthy hybrid wild boars from farms in Jilin,Inner Mongolia and Heilongjiang provinces.ELISA detected serum antibodies against classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),porcine circovirus type 2(PCV2)and Japanese encephalitis virus(JEV),but not against African swine fever virus(ASFV),with PCV2 having the highest seropositive rate(87.2–100%in different farms).RT-PCR or PCR performed on the processed samples detected only PCV2,with 33.1%(56/169)of blood samples and 32.8%(20/61)of spleen samples being positive,respectively,indicating widespread PCV2 infection in hybrid wild boars.Phylogenetic analysis of 15 PCV2 ORF2 sequences showed that they belong to genotypes PCV2a,PCV2b and PCV2d,with nucleotide and deduced amino acid homologies of 88.5–100%and 88.1–100%,respectively.

Analysis of the lifetime and culling reasons for AI boars

Background: The aim of the study was to analyze the lifetime and culling reasons for boars used in insemination centers(AI centers).Methods: The data collected from 355 culled boars from 1998 to 2013 included: age at start of semen collection,boar herd life, culling reason, daily gain and lean meat content, and number of ejaculates not meeting sales requirements after dilution. Culling reasons were divided into 7 groups: low semen value(LSV), low or lack of libido(LL), leg problems(LP), infectious diseases(ID), old age(OA), reduced demand for semen from the given boar(RD),and others(OT).Results: The most common culling reasons for boars were LSV(23.7%) and RD(22.5%). It was observed that the lowest daily gains were noted in boars culled due to OA. Boars culled due to OA and RD were maintained in production for the longest time(over 1000 d), for LSV and ID retention was about 700 d, and due to LL below 400 d. The survival probability was over 0.9 until 1.5 yr, and just over 0.2 until 4 yr. The highest relative frequency was observed in the 36 ^(th) and 42 ^(nd) mo of life(over 16%). Hazard risk analysis revealed a more than 10 times higher risk of culling in the case of LL, ID or OT, in comparison to OA.Conclusions: The results can be used as a direct point of reference for the identification of emerging problems in AI boar exploitation and the development of an appropriate culling policy in AI centers.

Semen characteristics of the three genetic types of boars reared in Benin

Objective:To characterize the semen of three genetic types of boars(local,improved and Large White)reared in Benin.Methods:Semen of local,improved and Large White boars reared in Benin were collected using the gloved hand method and analyzed to determine volume,pH,concentration,mobility,motility,and morphology.The effect of the genetic type of boar on semen characteristics was aslo studied.Results:Duration of ejaculation and semen volume of Large White boar were significantly higher than those of local and improved boars(P<0.05).The semen of improved boars had a higher motility score than that of Large White and local boars(P<0.001).The semen of local boars was more concentrated in the spermatozoa than that of improved and Large White boars(P<0.05).The proportion of spermatozoa of improved boars with normal morphology(93.6%)was significantly higher than that of local(82.2%)and Large White boars(81.6%)(P<0.001).The proportion of spermatozoa with folded tails in the semen of Large White boars(9.2%)was significantly higher than that observed in improved(1.8%)and local(5.0%)boars(P<0.001).The proportion of spermatozoa with proximal cytoplasmic droplets in semen of improved boars(2.7%)was significantly lower than that in Large White(6.8%)and local(9.7%)boars(P<0.001).The local(1.5%)and Large White boars(1.1%)showed more spermatozoa with distal cytoplasmic droplets in their semen compared to the improved boars(0.4%).Conclusions:The semen characteristics of pigs reared in Benin vary from one genetic type to another.Each genetic type has a strong point.The Large White boar produces larger semen,the local boar produces more concentrated semen and the improved boar produces spermatozoa that are morphologically better.The semen of these three genetic types can be used in artificial insemination but the improved boar's semen is more recommended.

Effect of Stabilized Fish Oil Source on Sperm Quality and Production of Boars

Research findings for supplementing boar stud diets with fish oils are inconsistent. This study was designed to address three possible causes of performance variation of boars to fish oil supplementation: stability of the fatty acid source, level of inclusion and breed of boars tested. Three groups of 87 boars each, from two genetic lines (PIC 337 and PIC 800), were assigned to treatment based on age, mean sperm production (previous 12 weeks), and body condition score. All boars received a corn-soybean meal diet with a commercial fish oil supplement providing 1.83 g/boar/day of docosahexaenoic acid (DHA) as a preconditioning diet. On 10-Aug., 2020, the DHA source was changed to a stabilized starch imbedded source of refined fish oil (Salmate®), providing 1.83 g/b/d for the test diet. Two additional levels providing 2.38 and 2.94 g/b/d of DHA were fed for a 9 week pretreatment period and during the test period. Salmate® fed at 2.38 g/b/d of DHA resulted in a reduction in the number of rejected ejaculates (P < 0.045) by 7.5% and 6.4% compared to the lowest and highest inclusion rates, respectively. There were no treatments by genetic line interactions. A retrospective study of semen production and quality of 77 boars on the Salmate® diet containing 1.83 g/b/d DHA was done to compare to the original source of DHA at the same inclusion level. There were no differences in semen quality parameters between the 2 lipid sources. Ejaculate volume increased from 177.9 ml to 233.4 ml (P < 0.001) and total sperm cells per ejaculate increased from 69.7 × 109 to 82.0 × 109 (P < 0.001) due to substitution of Salmate®. Adding Salmate® at 2.38 g/b/d resulted in a lower number of rejected ejaculates per boar by 7.5% and 6.4% vs. 1.83 and 2.94 g/b/d, respectively, and boars fed Salmate® at 1.83 g/b/d produced 17% more doses than the competing product.

Conflict between wild boars(Sus scrofa) and farmers:distribution, impacts,and suggestions for management of wild boars in the Three Gorges Reservoir Area

Economic losses caused by wild boars(Sus scrofa)continue to increase in the Three Gorges Reservoir Area.We selected 206 farmers in Wuqiaohe Basin as survey respondents using random stratified sampling.The distribution and impacts of wild boars were examined using a questionnaire survey and spatial analysis.The results showed that:(1)The density of farmer settlements is low and most peasants’households are located on the forest edge.Due to large-scale labor migration,a high proportion of farmland(25.85%)has been abandoned and an even higher proportion of farmland(40.48%)has been returned to forest.(2)The probability of wild boar damage to peasant households was 67.96%.Yields of corn and sweet potato decreased by 24.87%and 28.24%,respectively,with a total economic loss of approximately 2,590,100 RMB/yr in the Wuqiaohe basin.Sixty-five percent of the affected cultivatedlands were located within approximately 150 m of the forest edges.(3)The wild boar damage coefficient was significantly and negatively correlated with the distance between the peasants’household and the forest edge,the density of the settlements,and the area of cultivated land.The wild boar damage coefficient shows a significant positive correlation with the area of farmland returned to forest.(4)The damage activity caused by wild boars began to occur7-8 years ago,and the destruction has been more frequently and seriously observed in the last 4-5 years.(5)We make suggestions for wild boar control and management measures including developing a compensation policy.The compensation standard is related to the land area returning from farmland to forests and the actual cultivated land area.

Genetics Research and Advance on Development and Utilization of Wild Boars

Wild boar is one of the most important beast resources.It plays an important role in the maintenance of biological diversity.The genetic resources of wild boar can not only protect the genetic resources,but also improve the formation of new breeds in pigs.This paper summarized the advance on the main biological characteristics of wild boars,evolutionary origin between wild boars and domesticated pigs,and development and utilization of wild boars aimed to provide further insight into wild boar's genetic research and its resource protection.

Relationship between Serum L-Carnitine Levels and Sperm Parameters in Boars

This study evaluated the relationship between serum L-carnitine level and sperm parameters in young boars. Serum L-carnitine and semen characteristics were determined for 61 young Duroc boars between the ages of 590 and 630 days. Multiple linear regression analysis was performed to predict total and progressive motility and the total number of spermatozoa based on serum total L-carnitine and free L-carnitine levels. Total number of spermatozoa was not associated with basal serum L-carnitine levels. A regression equation was found in which both total L-carnitine levels and free L-carnitine levels were significant predictors of total and progressive motility (P 0.05). These results suggest that serum L-carnitine level is an important selection parameter for stock boars.

Epizootiological surveillance of porcine circoviruses in free-ranging wild boars in China

Four species of porcine circoviruses(PCV1–4)have been reported to circulate in Chinese domestic pigs,while the epizootiology of these viruses in free-ranging wild boars in China remains unknown.In this study,tissue and serum samples collected from diseased or apparently healthy wild boars between 2018 and 2020 in 19 regions of China were tested for the prevalence of PCV1–4 infections.Positive rates of PCV1,PCV2,and PCV3 DNA in the tissue samples of Chinese wild boars were 1.6%(4/247),58.3%(144/247),and 10.9%(27/247)respectively,with none positive for PCV4.Sequence analysis of viral genome showed that the four PCV1 strains distributed in Hunan and Inner Mongolia shared 97.5%–99.6%sequence identity with global distributed reference strains.Comparison of the ORF2 gene sequences showed that 80 PCV2 strains widely distributed in 18 regions shared 79.5%–100%sequence identity with reference strains from domestic pigs and wild boars,and were grouped into PCV2a(7),PCV2b(31)and PCV2d(42).For PCV3,17 sequenced strains shared 97.2%–100%nucleotide identity at the genomic level and could be divided into PCV3a(3),PCV3b(2)and PCV3c(12)based on the phylogeny of ORF2 gene sequences.Serological data revealed antibody positive rates against PCV1 and PCV2 of 11.4%(19/167)and 53.9%(90/167)respectively.The data obtained in this study improved our understanding about the epidemiological situations of PCVs infection in free-ranging wild boars in China and will be valuable for the prevention and control of diseases caused by PCVs infection.

Preliminary attempt to distinguish the domesticated pigs from wild boars by the methods of carbon and nitrogen stable isotope analysis

Despite great achievements in the origins of domestic pigs made by the methods of zooarchaeology and molecular biology,how to scientifically distinguish the domesticated pigs from wild boars during the early stage of pig domestication is still poorly understood.Compared to wild boar's diets which come from the natural environment,the diets of domestic pigs are more easily influenced by human feeding activities.Therefore,in principle,exploration of the dietary differences among pigs and understanding the impact on pig diets fed by humans can have great potential to differentiate between wild boars and domesticated pigs.To reveal dietary differences among pigs and distinguish the domesticated pigs from wild boars based on comparison with the diets of humans and other animals,we analyzed the carbon and nitrogen stable isotopes of human bones from Xiaojingshan Site and animal bones from Yuezhuang Site,both of which belong to Houli Culture in Shandong Province and date to about 8500―7500 years ago.The mean δ 13C value((-17.8 ± 0.3)‰) and δ 15N value((9.0±0.6)‰) in human collagen indicate that although millet agriculture began it was not the main subsistence strategy as millets are typical of C4 plants and that humans made a living mainly by gathering,hunting or raising some domesticated animals.The δ 13C value(-16.1‰) and δ 15N value(6.9‰) in the bovine suggest that C3 plants were dominant in its diet with some C4 plants complemented.The fish has lower δ 13C value(-24.9‰) and higher δ 15N value(8.8‰) than the bovine,which is the characteristic of the isotopic values from Eurasian freshwater fish.Based on the differences in carbon and nitrogen isotope values,the pigs can be divided into three groups.A group,composed of two pigs,has low δ 13C values(-18.1‰,-20.0‰) and low δ 15N values(4.7‰,6.0‰).B group,only one pig,has the highest δ 13C value(-10.6‰) and mediate δ 15N value(6.4‰).As for the C group,also only one pig,low δ 13C value(-19.0‰) and the highest δ 15N value(9.1‰) are observed.Previous studies on the stable isotopes from modern or ancient wild boars' bones have suggested that C3 plants are predominated in their diets and that their δ 15N values are close to those in herbivores and far from those in carnivores.Based on the comparison with the isotope values from humans,the wild boars and the domestic pigs from Xipo Site in Henan 6000―5500 years ago and Kangjia Site in Shaanxi 4500―4000 years ago,we conclude that A pig group belongs to wild boars while B and C groups can be attributed to domesticated pigs.

Glutamate and aspartate alleviate testicular/epididymal oxidative stress by supporting antioxidant enzymes and immune defense systems in boars

Several potential oxidative agents have damaging effects on mammalian reproductive systems.This study was conducted to investigate the effects of glutamate(Glu)and aspartate(Asp)supplementation on antioxidant enzymes and immune defense systems in the outer scrotum of boars injected with H2O2.A total of 24 healthy boars were randomly divided into 4 treatment groups:control(basal diet,saline-treated),H2O2(basal diet,H2O2-challenged outer scrotum(1 m L kg^–1 BW)),Glu(basal diet+2%Glu,H2O2-challenged),and Asp(basal diet+2%Asp,H2O2-challenged).Our results showed that both Glu and Asp supplementation improved testicular morphology and decreased the genital index in the H2O2-treated boars.Glu and Asp administration increased the antioxidant enzyme activities and affected the testicular inflammatory cytokine secretion but had no effect on sex hormone levels.Furthermore,the m RNA expression of CAT,Cu Zn SOD,and GPx4 was altered in the testes and epididymis of boars treated with Asp and Glu.Glu and Asp supplementation also modulated the expression of TGF-β1,IL-10,TNF-α,IL-6 and IL-1βin the testis and epididymis.These results indicate that dietary Glu and Asp supplementation might enhance antioxidant capacity and regulate the secretion and expression of inflammatory cytokines to protect the testes and epididymis of boars against oxidative stress.

Pig Reproductive and Respiratory Syndrome Virus (PRRSV) Does Not Attach to Boar Sperm;It Affects Only the Velocity Pattern and the Mobility Pattern

The purpose of the study was to evaluate the sperm viability of semen infected with PRRSV viral particles, observing the effect of the Virus on the motility of boar sperm. The work was carried out at the FMVZ-BUAP Genetics and Reproduction Laboratory. 5 stallions were used. Each sample contained 1 × 106 sperm, the PRRS virus strain was ATCC-VR-2332 (0, 102, 104 and 106 copies of RNA/mL in triplicate), it was observed daily at the CASA;Hamilton Thorne®. Cells with MT (P < 0.05) on days 1, 3, 5, 7 and 10 of evaluation with 201 ± 7.3, 167 ± 10.1, 165 ± 14.6, 134 ± 8.2 and 120 ± 8.8, respectively. The % MP between control and virus concentrations (P ≥ 0.05). The LCV on day 1 and 7 PI at 10X2 and 10X6 (P < 0.05) vs control. In the Correlation Matrix, where it is observed that there is a correlation between VSL and VAP, VSL and VCL, VCL and ALH, VAP with ALH. There is a correlation of VSL and ALH, STR and ALH. In this study there were (P ≤ 0.01) in the VCL, in the concentrations (102) 162.81 ± 10.65 and (106) 177.12 ± 5.77 vs 193.04 ± 4.62 of control. This indicates that altering these parameters would be related to fertility and the PRRS virus affects the LCV. Regarding the VSL, it was observed that the sperm infected with viruses 102, 104 and 106 of 48.00 ± 3.38, 49.88 ± 1.83 and 50.55 ± 2.24 Vs. 56.66 ± 1.68 of control respectively, the control would have greater possibilities of fertilizing the oocyte. In this study, it was found (P ≤ 0.01) in the VAP with 102 of 77.26 ± 5.16, 104 with 83.35 ± 2.41 and 106 with 81.29 ± 3.14 vs the control with 90.56 ± 2.07. Regarding the ALH there is (P < 0.05) a 104 with 8.70 ± .26 and 106 with 9.64 ± 0.23 vs control 8.50 ± 0.27. The presence of different concentrations of PRRSV in boar semen induces changes in different types of sperm motility. Infection of ejaculates with the PRRS virus affects sperm motility on days 1, 3, 5, 7, and 10 post-infections.

GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm.However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.

Nanotechnology-based approach for safer enrichment of semen with best spermatozoa

Background: Advances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles(MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic(annexin V) and acrosome-reacted(lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field(nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts(0, 87.5, and 175 μg) of MNP-conjugates(Annexin V-MNP or Lectin-MNP) and incubated(10 to15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated(0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates(87.5 μg– 30 min)on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.Results: Transmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved(P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between(P > 0.05).Conclusions: The findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable,and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

Background: The in vivo concentration of bicarbonate(HCO3-), one of the essential sperm capacitating effectors,varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3-concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased.Results: Once exposed to the capacitation medium, the intracellular pH(pHi) of spermatozoa increased immediately even at low concentrations of HCO3-, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation(pPKAs). Although with a significant delay, 15 mmol/L of HCO3-stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation(Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation(IVF)system based on the optimization of HCO3-concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes(8.6% in the standard system vs. 33.9%).Conclusions: Optimising HCO3-concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3-in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.

Study on the Accuracy of Different CASA Systems in the Quality Detection of Fresh Boar Semen

[Objectives]This study was conducted to compare and analyze the accuracy of computer-aided sperm analysis(CASA)and manual method for detecting the quality of fresh boar semen at room temperature.[Methods]Statistical methods such as analysis of variance,ZB score and Z score were used to compare the accuracy of five different brands of CASA systems and manual method to detect the vitality and density of fresh boar semen at room temperature.[Results]After setting the parameters of five CASA systems the same as follows:VCL(curvilinear velocity),VSL(straight-line velocity)≥5μm/s,STR(straightness)=VSL(straight-line velocity)/VAP(average path velocity)≥25%,the sperm motility of six parts of boar semen was tested at normal temperature using different brands of special fixed-volume slides with a uniform chamber height[(20±2)μm].There were no significant differences in sperm vitality detected by the five CASA systems(P>0.05).The ZB scores of the vitality obtained by observers or instrument engineers who did not have a job certificate from the quality inspection department showed that the results of three observers or instrument engineers were unsatisfactory(∣ZB∣>3),but there were no significant differences in vitality between the CASA systems and the inspector with a job certificate(P>0.05).Regarding sperm density detection,when the sperm density was less than 280×106/ml,there were no significant differences between the results displayed by the instruments and the results of the manual hemocytometer counting(P>0.05).[Conclusions]The accuracy of the CASA systems set to uniform parameters was consistent with the accuracy of the visual vitality obtained by an inspector with a job certificate.When the semen was diluted with 3%NaCl solution to a sperm density<280×10^6/ml,the sperm density detected by the CASA systems was consistent in reliability with that obtained by the hemocytometer detection.The CASA systems are faster and more efficient and objective than manual detection,and have the advantages of strong operability and easy promotion.

Flooding as a cause of ungulate mortality in floodplain forests in Croatia

Floodplain forests with regular flooding regimes are the largest natural retentions areas in Croatia and are important as natural habitats for ungulates.The aim of this study was to determine the scale of mortality caused by flooding within these forests.Over a 10-year period,data on ungulate mortality(red deer,roe deer and wild boar),flood duration and flooded surface area were recorded.The study was conducted in primary(Posavske Sume—RET I)and secondary(Opeke II—RET II)retention areas within Lonjsko Polje Nature Park(Sava River region,Croatia).The longest flood period and the largest flooded surface area were recorded in RET I.Total ungulate mortality was749 individuals,with 482 individuals in RET I and 267 individuals in RET II,predominantly wild boar.Flood mortality did not differ by gender.The highest mortality of wild boar was recorded for the juvenile and yearling ageclasses.Low mortality of red and roe deer can be attributed to their body size and ecological niches.Differences in mortality between the primary and secondary retention areas corresponded to differences in flood regimes,flood column heights and micro relief structures.In both retention areas,wild boar mortality and flood duration,i.e.flooded surface area,were positively correlated.Because the growth rate of the analysed ungulate populations was higher than the recorded mortality,no long-term effect of floods is expected on species abundance in these areas.

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super AQPs.AQP3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation.Water exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of sperm.However,the freezethaw process leads to sperm damage and a loss of fertilizing potential.Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen freezability.In this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results:Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability.Moreover,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

In vitro performance and in vivo fertility of antibiotic-free preserved boar semen stored at 5℃

Background: Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility.Objectives: To examine chilling injury and the number and type of bacteria in boar semen stored at 5 ℃ in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions.Material and methods: Boar ejaculates were extended with AndroStar~? Premium, stored at 17 ℃ with and at 5 ℃ without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study.Results: Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher(P < 0.05) up until 72 h in sperm stored at 5 ℃ compared to 17 ℃ but did not differ after 144 h.After 72 h, incubation in capacitating medium for 60 min induced a similar decrease in viable sperm with high mitochondria membrane potential and low cytosolic calcium in both groups. In semen stored at 5 ℃, bacteria counts were below 103 CFU/mL and the bacteria spectrum was similar to that of raw semen. In 88% of 34 boars,cooled semen fulfilled the requirements for insemination. Fertility was high and did not differ(P > 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 ℃ and semen stored at 17 ℃ with antibiotics.Conclusion: Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.

Identification of Toll-like Receptor (TLR) 4 Gene in Wild Boar

Toll-like receptor(TLR)4 plays an important role in the innate immune system and has been involved in resistance/susceptibility to a number of diseases as revealed by studies in human and other domestic animals.Wild boar survives in natural environment without artificial interference and may be different from domestic pig in innate immune system.Here,the complete coding sequence of TLR4 and TLR4A was cloned in wild boar,and two other alternative splicing variants,TLR4B and TLR4C,were obtained.Compared to the counterpart from domestic pig(Gen Bank No.AJ628065),there were five SNPs,c.510T>C,c.960A>G,c.962A>G,c.1605T>G and c.1824A>G,in the coding sequence of wild boar TLR4A gene.TLR4 gene was expressed in all the tissues from wild boar studied with the most abundance in spleen tissue,and m RNA level was significantly lower in spleen from wild boar than that from Min pig.The allele distribution was significantly different at polymorphic loci c.962G>A and c.1027C>A(p<0.01)between wild boar and Min pig.The results would contribute to understand the innate immune system in wild boar.