Rumen microbial degradation of bromoform from red seaweed(Asparagopsis taxiformis)and the impact on rumen fermentation and methanogenic archaea

Background The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane(CH_(4))analogues,primarily bromoform(CHBr_(3)).This study aimed to investigate the degradation process of CHBr3 from A taxiformis in the rumen and whether this process is diet-dependent.An in vitro batch culture system was used according to a 2×2 factorial design,assessing two A taxiformis inclusion rates[0(CTL)and 2%DM diet(AT)]and two diets[high-concentrate(HC)and high-forage diet(HF)].Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0,0.5,1,3,6,8,12,16,24,48 and 72 h to assess the pattern of degradation of CHBr_(3) into dibromomethane(CH_(2)Br_(2))and fermentation parameters.Additionally,an in vitro experiment with pure cultures of seven methanogens strains(Methanobrevibacter smithii,Methanobrevibacter ruminantium,Methanosphaera stadtmanae,Methanosarcina barkeri,Methanobrevibacter millerae,Methanorhermobacter wolfei and Methanobacterium mobile)was conducted to test the effects of increasing concentrations of CHBr3(0.4,2,10and 50μmol/L).Results The addition of AT significantly decreased CH_(4) production(P=0.002)and the acetate:propionate ratio(P=0.003)during a 72-h incubation.The concentrations of CHBr_(3) showed a rapid decrease with nearly 90%degraded within the first 3 h of incubation.On the contrary,CH_(2)Br_(2) concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation.Neither CHBr_(3) degradation nor CH_(2)Br_(2) synthesis were affected by the type of diet used as substrate,suggesting that the fermentation rate is not a driving factor involved in CHBr_(3)degradation.The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M.smithii,M.ruminantium,M.stadtmanae,M.barkeri,M.millerae,M.wolfei,and M.mobile.Conclusions The present work demonstrated that CHBr_(3) from A.taxiformis is quickly degraded to CH_(2)Br_(2)in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr_(3)degradation.

Suppressive effects of acetone extract from the stem bark of three Acacia species on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species(Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production.Methods: The lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase,cyclooxygenase-2 and tumor necrosis factor-a. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spectrometry/mass spectrometry analysis.Results: All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-a in a concentration dependent manner(25, 50 and 75 mg/m L). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components.Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.

Hepatic Expression of Insulin-Like Growth Factor-1 inUnderfed Pregnant Ewes

The liver is one of the most important visceral organs, which represents a large contribution to whole animal energyexpenditure and the major synthetic site of insulin-like growth factor-I （IGF-1） peptide. Decreased plane of nutrition acts byreducing the metabolic rate and mass of metabolic tissues, such as liver. Also, undernutrition results in the reduced circulating IGF-1concentrations, due to the uncoupled growth hormone-IGF （GH-IGF） axis. This study investigated whether a 22-day period ofundernutrition （half maintenance） could affect liver mass and IGF-1 protein and gene expression. Sixteen pregnant ewes fed all （n =9） or half （n = 7） of their maintenance energy requirements were slaughtered on day 7 of pregnancy （oestrus = day 0）. Body and livermass, IGF-1 plasmatic concentrations and liver IGF-1 mRNA and protein expression were determined. Liver mass and the proportionof liver mass to empty body weight were lower in underfed animals. While IGF-1 plasmatic concentrations were lower inundernourished ewes, no differences in liver mRNA expression were found. This is the first time that differences inimmunohistochemistry intensity and total content are reported in sheep. In summary, the decreased plasma IGF-I concentrationsinduced by undernutrition in ewes was not associated with its reduced hepatic mRNA or protein expression, but to a decrease in livermass.

Key Factors Involved in Lamb Quality from Farm to Fork in Europe

World lamb consumption is approximately 2 kg per capita with large variations between continents, e.g., 17 kg in Oceania vs. 0.7 kg in North America. With less than one million tonnes, the international trade of sheep meat contributes to a small percentage of the total meat exchanged between countries. On the other hand, lamb represents the highest rate of species trading in relation to the total sheep meat produced. It is therefore likely to ifnd a wide variability of different lamb products in the same market, and that the conservation procedures （such as refrigeration, packaging or freezing）, chemical composition, especially the fat due to its implication in human health, lfavour development and juiciness perception （affected, i.e., by the feeding or the age or slaughter weight of the animal） and acceptability （based on culinary background） could be considered important indicators of this advantageous situation. In this paper some studies related to the aforementioned indicators are discussed.

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3-5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis （CASA-Mot）, and of sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations （slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]）, and three morphometric subpopulations （large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]）. The distribution of kinematic sperm subpopulations was different among ejaculate fractions （P 〈 0.001）, with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions （P〈 0.001）, with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin （PI/PSA） combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull 〉 ram 〉 boar. However, for the other morphometric parameters （length, width, and perimeter）, there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.

Relationship of sperm plasma membrane andacrosomal integrities with sperm morphometry inBos taurus

To date,sperm morphometric studies have assessed whole sperm populations without considering sperm function.The aim of this study was to evaluate the relati on ship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls.To this end,sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes,including Hoechst 33343,carboxyfluorescein diacetate,and propidium iodide.This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometries of the sperm head,nucleus,and acrosome using a specific plug-in module created on ImageJ.Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome(P<0.001).In the case of spermatozoa with an intact acrosome,those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane(P<0.001).No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations.There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for in tact spermatozoa.These correlations were particularly strong for the morphometric parameters of the sperm acrosome.We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

Morphometry and subpopulation structure of Holstein bull spermatozoa： variations in ejaculates and cryopreservation straws

Sperm quality is evaluated for the calculation of sperm dosage in artificial reproductive programs. The most common parameter used is motility, but morphology has a higher potential as a predictor of genetic quality. Morphometry calculations from CASA-Morph technology improve morphological evaluation and allow mathematical approaches to the problem. Semen from 28 Holstein bulls was collected by artificial vagina, and several ejaculates were studied. After general evaluation, samples were diluted, packaged in 0.25 ml straws, and stored in liquid nitrogen. Two straws per sample were thawed, and slides were processed and stained with Diff-Quik. Samples were analyzed by a CASA-Morph system for eight morphometric parameters. In addition to the ＂classical＂ statistical approach, based on variance analysis （revealing differences between animals, ejaculates, and straws）, principal component （PC） analysis showed that the variables were grouped into PC1, related to size, and PC2 to shape. Subpopulation structure analysis showed four groups, namely, big, small, short, and narrow from their dominant characteristics, representing 31.0%, 27.3%, 24.1%, and 17.7% of the total population, respectively. The distributions varied between animals and ejaculates, but between straws, there were no differences in only four animals. This modern approach of considering an ejaculate sperm population as divided into subpopulations reflecting quantifiable parameters generated by CASA-Morph systems technology opens a new view on sperm function. This is the first study applying this approach to evaluate different ejaculates and straws from the same individual. More work must be done to improve seminal dose calculations in assisted reproductive programs.

Afterword to Sperm morphometrics today and tomorrow special issue in Asian Journal of Andrology

The problems associated with the subjective assessment of human sperm morphology have been well aired in another Asian Journal of Andrology Special Issue1 that marked the publication of the 5th edition of the WHO Semen analysis manual, and contrary views have subsequently been presented） However, the vagaries of the eye-brain system in assessing whether a sperm head is large or small can be eliminated by objective assessment where definitive structures are defined by their dimensions. These can then be classified automatically into as many categories as the data permit, conventionally on the basis of preset upper and lower limits, but also by more comprehensive analysis as discussed here.

Bioactive Low Molecular Weight Compounds in Two Traditional Spanish Products

The aim of this work focuses on the study of low molecular weight (LMW) compounds with antioxidant and antihy-pertensive effect in Iberian ham and La Serena cheese, two traditional products in Spain, in comparison to cured ham and matured Ewe’s milk cheese. Proteolysis parameters were studied, LMW compounds were isolated and their antioxidant and antihypertensive activity was analyzed. Results showed that a more intense proteolysis, expressed as peptidic nitrogen (PN), in Iberian ham (p < 0.001), can cause higher antioxidant activity in this product for both DPPH radical scavenging (p < 0.001) and metal chelating effect (p < 0.001). However, a more intense proteolysis in La Serena cheese, expressed as non protein nitrogen (NPN) (p < 0.001) and PN (p < 0.001), did not promote higher antioxidant activity in this cheese. On the other hand, no differences were found for antihypertensive activity, expressed as angiotensin I-converting enzyme (ACE) inhibitory activity, in both type of hams or cheese.

Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three morphometric subpopulations （large-round 30.4%, small-round 46.6%, and large-elongated 22.9%）. In the second analysis, using discriminant methods, the classification was made independently of size and shape. Three morphological categories according to nuclear size （small 〈10.90 μm^2, intermediate 10.91-13.07 μm^2, and large 〉13.07 μm^2） and four categories were defined on 400 canonical cells （100 × 4） from 10 men according to sperm nuclear shape （oval, pyriform, round, and elongated）. Thereafter, the resulting classification functions were used to categorize 4200 spermatozoa from 21 men. Differences in the class distribution were observed among men from both clustering and discriminant procedures. It was concluded that the combination of CASA-Morph fluorescence-based technology with multivariate cluster or discriminant analyses provides new information on the description of different morphometric sperm subpopulations in normal individuals, and that important variations in the distribution of morphometric sperm subpopulations may exist between men, with possible functional implications.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex

This study was designed to determine the ability of computer-assisted sperm morphometry analysis （CASA-Morph） with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa （SX and SY, respectively）. Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average （P 〈 0.001） although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed （mixed SX and SY） and sexed （SX） semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa （P 〈 0.05）. We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.