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The improvement of carcass quality is one of the main breeding goals in pig production. To select appropriate breeding animals, it is of major concern to exactly and reliably analyze the body composition in vivo. Therefore, the objective of the study was to examine whether the combination of dual-energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI) offers the opportunity to reliably analyze quantitative and qualitative body composition characteristics of different pig breeding groups in vivo. In this study, a total of 77 pigs were studied by DXA and MRI at an average age of 154 days. The pigs originated from different autochthonous or conventional breeds or crossbreeds and were grouped into six breed types: Cerdo Ibérico (Ib); Duroc × Ib (Du_Ib); White Sow Lines (WSL, including German Landrace and German Large White); Hampshire/Pietrain (Pi_Ha, including Hampshire, Pietrain × Hampshire (PiHa) and Pietrain × PiHa); Pietrain/Duroc (Pi_Du, including Pietrain × Duroc (PiDu) and Pietrain × PiDu); crossbred WSL (PiDu_WSL, including Pietrain × WSL and PiDu × WSL). A whole-body scan was performed by DXA with a GE Lunar DPX-IQ in order to measure the amount and percentage of fat tissue (FM; %FM), lean tissue (LM; %LM) and bone mineral, whereas a Siemens Magnetom Open with a large body coil was used for MRI in the thorax region between 13th and 14th vertebrae in order to measure the area of the loin (LA) and the above back fat area (FA) of both body sides. A GLM procedure using SAS 9.2 was used to analyze the data. As expected, the native breed Ib followed by Du_Ib crossbreeds showed the highest %FM (27.2%, 25.0%) combined with the smallest LA (46.2 cm2, 73.6 cm2), whereas Ib had the lowest BW at an average age of 154 days. Pigs with Pi_Ha origin presented the least %FM (12.4%) and largest LA (99.5 cm2). The WSL and PiDu_WSL showed an intermediate body composition. Therefore, it could be concluded that DXA and MRI and especially their combination are very suitable methods to reliably identify differences in body composition and carcass traits among different pig lines in vivo.
The human–animal relationship is an important component of the welfare of farm animals and for this reason animal responsiveness tests to humans are included in on-farm welfare assessment schemes that provide indicators for this. However, apart from the behaviour of stockpersons towards their animals, other factors may also influence animals’ reactivity to humans as observed through behavioural tests, which can add a further layer of complexity to the interpretation of test results. Knowledge of these factors may help a better interpretation of differences from one farm to another in the outcome of human–animal relationship tests, and may provide clues for improving the relationship between animals and humans. The main objective of this study was to identify whether management or environmental factors could influence the outcome of human–animal relationship tests in veal calves. Two tests were performed when calves were aged 14.9 ± 1.6 (SD) weeks in 148 veal farms: the voluntary approach of an unfamiliar human standing at the feeding fence and the reaction towards an unfamiliar human who entered the home pen and tried to touch each calf in a standardised way (Calf Escape Test (CET) – score 0 to 4). Questionnaires were filled in and interviews with the stockpersons were performed in order to obtain information on stockpersons, management, animal and building characteristics. The latency to touch an unfamiliar human at the feeding fence was significantly correlated with the CET scores. Total number of calves on the farm, space allowance, breed, environmental enrichment, stockperson's experience and season of observation influenced the percentage of calves that scored 0 in CET (i.e. calves that could not be approached). Type of milk distribution, type of breed and number of calves per stockperson influenced the percentage of calves that scored 4 in CET (i.e. calves could be touched). For both CET0 and CET4, the level of self-reported contacts by the stockperson (analysed only on the French subset of 36 farms) did not influence the results. This paper concludes that according to the tests conducted on veal calves on commercial farms, factors such as milk distribution method, breed of the calves or the level of experience of stockpersons with veal farming can have an impact on the results of tests focusing on human–animal relationships.
This research investigated two sources of sire-specific genetic effects on the birth weight (BWT) and weaning weight (WWT) of Bruna dels Pirineus beef calves. More specifically, we focused on the influence of genes located in the non-autosomal region of the Y chromosome and the contribution of paternal imprinting. Our analyses were performed on 8130 BWT and 1245 WWT records from 12 and 2 purebred herds, respectively, they being collected between years 1986 and 2010. All animals included in the study were registered in the Yield Recording Scheme of the Bruna dels Pirineus breed. Both BWT and WWT were analyzed using a univariate linear animal model, and the relevance of paternal imprinting and Y chromosome-linked effects were checked by the deviance information criterion (DIC). In addition to sire-specific and direct genetic effects, our model accounted for random permanent effects (dam and herd-year-season) and three systematic sources of variation, that is, sex of the calf (male or female), age of the dam at calving (six levels) and birth type (single or twin). Both weight traits evidenced remarkable effects from the Y chromosome, whereas paternal imprinting was only revealed in WWT. Note that differences in DIC between the preferred model and the remaining ones exceed 39 000 and 2 800 000 DIC units for BWT and WWT, respectively. It is important to highlight that Y chromosome accounted for ∼2% and ∼6% of the total phenotypic variance for BWT and WWT, respectively, and paternal imprinting accounted for ∼13% of the phenotypic variance for WWT. These results revealed two relevant sources of sire-specific genetic variability with potential contributions to the current breeding scheme of the Bruna dels Pirineus beef cattle breed; moreover, these sire-specific effects could be included in other beef cattle breeding programs or, at least, they must be considered and appropriately analyzed.
Injection anaesthesia with a combination of ketamine and azaperone (K/A) is discussed as a painless alternative to commonly used non-anaesthetized castration. To protect anaesthetized piglets from being crushed, they have to be separated from the sow for 3 h following castration. The aim of this study was to test if this separation and the different treatments would affect short-term behaviour after castration (3 to 6 h after castration) as well as weight gain. Piglets were 5 to 7 days old. Treatment Group 1 received a combination of anaesthesia and analgesia (n = 29, ketamine: 25 mg/kg BW; azaperone: 2 mg/kg BW; meloxicam: 0.4 mg/kg BW), Group 2 received only analgesia (n = 24) and Group 3 received no medication (n = 29). Behaviour and suckling order were compared for a 3 h period the day before castration and after castration. A significantly higher number of teats used by anaesthetized piglets (P = 0.004) suggests a decrease in suckling order stability. There were significant treatment effects between all three groups in the time spent at the sow's teat, with an increase in Group 2 (+69%), decrease in Group 1 (−28%), whereas the control Group 3 (+2%) almost remained unchanged. The anaesthetized piglets showed an increase in the time spent active away from the sow after castration of almost 200% (Groups 2 and 3: ∼50%, P < 0.001). However, no significant treatment effect was seen for weight gain. The results suggest that analgesia has an effect on behaviour, perhaps due to less post-castration pain. This advantage is not apparent for animals receiving additional anaesthesia, probably because of impaired coordination. Although the behavioural changes did not affect weight gain significantly, a decrease in suckling order stability indicates a certain degree of stress due to fighting over teat positions as a consequence of separation. Thus, post-castration behaviour must be taken into account when evaluating alternative castration methods.
Pigs are confronted with changes in farming practices that may affect performance and animal well-being. The sanitary conditions of the farm can have an impact on the ability of pigs to adapt to these changes. This study aimed to analyse how weaned pigs respond to common farming practices of changes in diet and housing in terms of performance, health and behaviour, and how these responses are affected by the sanitary housing conditions, qualified here as good or poor. At weaning at 4 weeks of age, 20 piglets were assigned to 10 blocks of two littermates and each pig within a litter was randomly assigned to one of two sanitary conditions. Pigs were housed individually and received a starter diet. A diet change occurred on day 12 post weaning (starter to weaner diets) and pigs were transferred to the grower unit on day 33 post weaning and continued to receive the weaner diet. From 43 days post weaning, pigs were offered a grower diet and were vaccinated against swine influenza on day 47 and 61 post weaning. On the basis of this design, three post-weaning phases were identified: phase I from day 1 to 11 (post weaning), phase II from day 12 to 32 (after the diet change) and phase III from day 33 to 42 (after the housing change). Individual BW was measured every 3 days, and feed refusals and faecal scores were recorded on a daily basis. Behavioural observations were performed during 28 days by using the instantaneous scan sampling method. Individual blood samples were collected at the end of each phase to analyse the plasma concentration of haptoglobin and on day 68 post weaning to analyse the anti-influenza immunoglobulins G (IgG). Poor sanitary conditions resulted in a decrease in daily gain, feed intake and gain to feed ratio of, respectively, 11%, 5% and 7% (P < 0.05). Pigs in poor sanitary conditions had higher faecal scores (P < 0.05), tended to have higher plasma haptoglobin concentration in phase II (P = 0.06) and had a higher anti-influenza IgG titre (P = 0.11). The diet change affected performance and behavioural responses of pigs in poor but not in good sanitary conditions. Housing change resulted in a 30% decrease in growth and an increase in behaviour oriented towards exploration and excitement. The results of this study show an effect of sanitary conditions on the responses of pigs to a diet change, whereas those to a housing change were little affected by the sanitary conditions.
Once daily milking (ODM) is a management practice that can improve working conditions and reduce production costs in dairy farming compared with twice daily milking (TDM). However, ODM is associated with a decrease in milk yield. Previous research indicated that disruption of tight junctions in the mammary gland may be one of the regulatory factors involved in the milk yield decrease observed during ODM. The aim of this study was to investigate the involvement of mammary epithelium disruption in the regulation of the activity and dynamics of mammary epithelial cells (MEC) during 5 weeks of ODM in goats. Twelve alpine goats (producing 3.67 ± 0.64 kg/day and 47 ± 1.6 days in milk) were assigned to two groups that were milked once or twice a day during 5 weeks and then switched back to TDM. Mammary biopsies were collected before and on days 2 and 16 of both ODM and TDM switchback periods. Milk purified epithelial cells were collected before and on days 1, 7, 21 and 28 during ODM as well on days 1 and 7 of the TDM switchback period. The mRNA levels of genes involved in the regulation of synthetic activity and apoptosis were analysed by RT-PCR in milk MEC and mammary biopsies. ODM decreased yields of milk (−23%), lactose (−23%) and casein (−16%). Lactose synthesis was regulated at the transcriptional level by downregulation of α-lactalbumin mRNA levels in both biopsy samples (−30%) and milk MEC (−74%). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining of mammary gland biopsies did not show any increase in cell apoptosis after 2 and 16 days of ODM (0.8% and 1%, respectively) despite upregulation of Bax mRNA levels in milk MEC. This suggests that the decrease in milk yield observed during ODM is attributable to a decrease in synthetic activity rather than to induction of MEC cell death. ODM induced the disruption of tight junctions in the mammary gland only on the first day of the treatment as indicated by increased blood lactose concentration. This indicates that the decrease in MEC activity observed over the 5 weeks of ODM was not due to disruption of the mammary gland tight junctions. There was no carryover effect of 5 weeks of ODM on milk production. Therefore, it appears that the decrease in milk yield that occurs during ODM in goats is due to regulation of synthetic activity rather than to apoptosis of MEC.
This study aimed to assess whether inclusion of chicory or lupine (prebiotics) in the diet of pre-slaughter pigs for just 1 or 2 weeks could change the composition of their intestinal microbiota, stimulate the growth of bifidobacteria and help to lower the amount of thermoplilic Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli), which are a major cause of food-borne infections in humans. A total of 48 pigs that had an initial live weight of 90 kg were fed with either a lupine (organic concentrate with 25% blue lupine seeds), chicory (organic concentrate with 10% dried chicory roots) or control (100% organic concentrate) diet for 1 week (24 pigs) or 2 weeks (24 pigs) before slaughter. The Campylobacter spp. level in rectal faecal samples after 0, 1 and 2 weeks of feeding and in the luminal content from ileum, caecum and colon at slaughter was determined by direct plating on modified charcoal-cefoperazone-deoxycholate agar plates. DNA extracted from the luminal content of distal ileum and caecum was used for terminal restriction fragment length polymorphism (T-RFLP) analysis of the composition of intestinal microbiota and for measuring the amount of bifidobacterial and total bacterial DNA by quantitative real-time PCR (qPCR). Campylobacter spp. were excreted by all pigs and present in the luminal content from distal ileum to midway colon with particularly high numbers in the caecum, but the excretion was reduced by 10-fold in pigs fed lupines for 1 week as compared with control- and chicory-fed pigs (mean log10 2.9 v. 4.1 CFU/g; P < 0.05). The qPCR analysis showed that feeding with lupines resulted in higher levels of bifidobacteria in caecum as compared with the other diets (P < 0.05). T-RFLP analysis showed that four of the most abundant bacteria with terminal restriction fragment values >5% relative to the intensity of total abundance differed between the feed treatments (P < 0.05). Therefore, this study showed that even a short-term alternative feeding strategy with prebiotics in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine increased the level of bifidobacteria in caecum and reduced the Campylobacter spp. excretion level after 1 week.
The aim of this study was to investigate the accuracy to predict detailed fatty acid (FA) composition of bovine milk by mid-infrared spectrometry, for a cattle population that partly differed in terms of country, breed and methodology used to measure actual FA composition compared with the calibration data set. Calibration equations for predicting FA composition using mid-infrared spectrometry were developed in the European project RobustMilk and based on 1236 milk samples from multiple cattle breeds from Ireland, Scotland and the Walloon Region of Belgium. The validation data set contained 190 milk samples from cows in the Netherlands across four breeds: Dutch Friesian, Meuse-Rhine-Yssel, Groningen White Headed (GWH) and Jersey (JER). The FA measurements were performed using gas–liquid partition chromatography (GC) as the gold standard. Some FAs and groups of FAs were not considered because of differences in definition, as the capillary column of the GC was not the same as used to develop the calibration equations. Differences in performance of the calibration equations between breeds were mainly found by evaluating the standard error of validation and the average prediction error. In general, for the GWH breed the smallest differences were found between predicted and reference GC values and least variation in prediction errors, whereas for JER the largest differences were found between predicted and reference GC values and most variation in prediction errors. For the individual FAs 4:0, 6:0, 8:0, 10:0, 12:0, 14:0 and 16:0 and the groups’ saturated FAs, short-chain FAs and medium-chain FAs, predictions assessed for all breeds together were highly accurate (validation R2 > 0.80) with limited bias. For the individual FAs cis-14:1, cis-16:1 and 18:0, the calibration equations were moderately accurate (R2 in the range of 0.60 to 0.80) and for the individual FA 17:0 predictions were less accurate (R2 < 0.60) with considerable bias. FA concentrations in the validation data set of our study were generally higher than those in the calibration data. This difference in the range of FA concentrations, mainly due to breed differences in our study, can cause lower accuracy. In conclusion, the RobustMilk calibration equations can be used to predict most FAs in milk from the four breeds in the Netherlands with only a minor loss of accuracy.
Two experiments were conducted to evaluate the effects of Bacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 1011 colony-forming units (cfu) of B. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 1011 cfu of B. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 1011 and 1.0 × 1011 cfu of B. subtilis natto linearly increased (P < 0.01) milk production (25.2 and 26.4 kg/day v. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/day v. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/day v. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/day v. 0.88 kg/day), protein (0.77 and 0.82 kg/day v. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/day v. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P < 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 1011 cfu/day B. subtilis natto from days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21. B. subtilis natto was discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P < 0.01), whereas ammonia nitrogen (NH3-N), total volatile fatty acids and molar proportion of propionate (P < 0.01) and valerate (P < 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P < 0.01) during the trial period. However, no differences for 24-h in sacco dry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P < 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P < 0.01) but protozoa decreased to 5.35 log10 cfu/ml (P < 0.01) during the trial period. These results demonstrate that B. subtilis natto improves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate that B. subtilis natto has potential to be applied as a probiotic for dairy cows.
Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic–pituitary–gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.
Genome-wide association studies for difficult-to-measure traits are generally limited by the sample size with accurate phenotypic data. The objective of this study was to utilise data on primiparous Holstein–Friesian cows from experimental farms in Ireland, the United Kingdom, the Netherlands and Sweden to identify genomic regions associated with the feed utilisation complex: fat and protein corrected milk yield (FPCM), dry matter intake (DMI), body condition score (BCS) and live-weight (LW). Phenotypic data and 37 590 single nucleotide polymorphisms (SNPs) were available on up to 1629 animals. Genetic parameters of the traits were estimated using a linear animal model with pedigree information, and univariate genome-wide association analyses were undertaken using Bayesian stochastic search variable selection performed using Gibbs sampling. The variation in the phenotypes explained by the SNPs on each chromosome was related to the size of the chromosome and was relatively consistent for each trait with the possible exceptions of BTA4 for BCS, BTA7, BTA13, BTA14, BTA18 for LW and BTA27 for DMI. For LW, BCS, DMI and FPCM, 266, 178, 206 and 254 SNPs had a Bayes factor >3, respectively. Olfactory genes and genes involved in the sensory smell process were overrepresented in a 500 kbp window around the significant SNPs. Potential candidate genes were involved with functions linked to insulin, epidermal growth factor and tryptophan.
Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate – as potent specific inhibitors of protein phosphatase calcineurin – can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin – cyclosporin A or hymenistatin I – on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin – allethrin or permethrin – had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.
Sirtuins are NAD+-dependent histone and protein deacetylases, which have been studied during the last decade with a focus on their role in lifespan extension and age-related diseases under normal and calorie-restricted or pathological conditions. However, sirtuins also have the ability to regulate energy homeostasis as they can sense the metabolic state of the cell through the NAD+/NADH ratio; hence, changes in the diet can modify the expression of these enzymes. Dietary manipulations are a common practice currently being used in livestock production with favorable results, probably due in part to the enhanced activity of sirtuins. Nevertheless, sirtuin expression in livestock species has not been a research target. For these reasons, the goal of this review is to awaken interest in these enzymes for future detailed characterization in livestock species by presenting a general introduction to what sirtuins are, how they work and what is known about their role in livestock.
In dairy cows, the intensity of metabolic activity, associated with the negative energy balance (NEBAL), is responsible for an increased production of reactive oxygen species (ROS) and, subsequently, for the development of the condition of oxidative stress, which may overwhelm the antioxidant potential of the bovine maternal organism, making it prone to the development of many puerperal dysfunctions, as well as to an alteration of colostrum and milk quality. Given these premises, the aims of this study are to evaluate serum and milk concentrations of ROS and lipoperoxides, vitamins A and E, on the 10th, 12th, 14th and 16th day postpartum of dairy cows, a particularly critical period during which the NEBAL reaches its nadir, and to compare the trends of these parameters in two different bovine breeds. The study was performed in pluriparous Italian Friesian and Brown dairy cows. On the 10th day postpartum, all cows underwent a clinical examination to exclude the presence of alterations; furthermore, on the same day, a milk sample was collected from each cow, in order to perform the somatic cell count (SCC; (CE) N. 853/2004) and to establish which of them had an SCC ⩽400 000/ml or >400 000/ml. In this study, among the 110 cows that were initially selected, the evaluation of these parameters allowed the inclusion of 80 animals, which were divided into four groups of 20 subjects each: Group F and F1: Italian Friesian healthy cows, with SCC ⩽400 000/ml and >400 000/ml, respectively; Group B and B1: Italian Brown healthy cows, with SCC ⩽400 000/ml and >400 000/ml, respectively. On the 10th, 12th, 14th and 16th day postpartum, peripheral blood and milk samples were collected. The results obtained show that in group B1 there were higher concentrations of ROS and milk antioxidants compared with Friesian group cows. This datum let us suppose that even in the presence of higher ROS concentrations the antioxidant status found in group B1 seems to be able to counteract the oxidative damage, which is more likely to develop in these cows.
Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP–DPAM, SNAP–OAG and SNAP–PMA groups). After invitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP–OAG 0, SNAP–DPAM 17.4 ± 2.5% and SNAP–PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca2+ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP–DPAM 20.7 ± 2.6%, SNAP–OAG 16.7 ± 2.9% or SNAP–PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.
The aim of this study was to investigate whether the use of sainfoin-based condensed tannins (CT) enhances feed value when given with tannin-free legumes (lucerne) to sheep. The experiments were conducted with fresh sainfoin and lucerne harvested at two stages (vegetative stage as compared with early flowering) in the first growth cycle. Fresh sainfoin and lucerne forages were combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (denoted S100, S75, S25 and S0, respectively). Voluntary intake, organic matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed the different sainfoin and lucerne mixtures. Loss of dry matter (DM) and N from polyester bags suspended in the rumen, abomasum and small intestine (SI) was also measured using rumen-fistulated sheep and intestinally fistulated sheep. The CT content in sainfoin (S100) decreased with increasing percentage of lucerne in the mixture (mean value from 58 g/kg DM for S100 to 18 g/kg DM for S25) and with growth stage (S100: 64 to 52 g/kg DM). OMD did not differ between different sainfoin/lucerne mixture ratios. Sainfoin and lucerne had an associative effect (significant quadratic contrast) on voluntary intake, N intake, total-tract N digestibility, N in faeces and urine (g/g N intake) and N retained (g/g N intake). Compared with lucerne mixtures (S0 and S25), high-sainfoin-content mixtures (S100 and S75) increased the in situ estimates of forage N escaping from the rumen (from 0.162, 0.188 for S0 and S25 to 0.257, 0.287 for S75 and S100) but decreased forage N intestinal digestibility (from 0.496, 0.446 for S0 and S25 to 0.469, 0.335 for S75 and S100). The amount of forage N disappearing from the bags in the SI (per g forage N) was the highest for high-sainfoin mixtures (from 0.082, 0.108 for S100 and S75 to 0.056, 0.058 for S25 and S0, P < 0.001). Rumen juice total N (tN) and ammonia N (NH3-N) values were the lowest in the high-sainfoin diet (mean tN 0.166 mg/g in S100 as compared with 0.514 mg/g in S0; mean NH3-N 0.104 mg/g in S100 as compared with 0.333 mg/g in S0, P < 0.001).